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An Rgd-modified Endostatin Peptide Expressed at e. Coli shows Anti-tumor Activity in vivo
Hai Tao Zhang,  Hui Cheng Li,   Zheng Wu Li,  ChangHong Guo and Yu Jun Chen
[Abstract] [FULL-TEXT INQUIRY] [PMID: 21675950 PubMed - indexed for MEDLINE] [BSP/PPL/E-Pub/00349]


Predicting Viral Protein Subcellular Localization with Chou’s Pseudo Amino Acid Composition and Imbalance-Weighted Multi-Label K-Nearest Neighbor Algorithm
Jun-Zhe Cao and Hong Gu
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00431]


Incorporating Secondary Features Into The General Form Of Chou’s Pseaac For Predicting Protein Structural Class
Bo Liao,Qilin Xiang and Dachao Li
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00432]


Epitope Mapping And Identification Of Amino Acids Critical For Rabbits Igg-Binding To Linear Epitopes On Buffalo Beta-Lactoglobulin
Li Xin, Chen Hongbing, Gao Jinyan, Liu Fahui and Wen Xuefang
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00446]


Identify Gpcrs And Their Types With Chou’s Pseudo Amino Acid Composition: An Approach From Multi-Scale Energy Representation And Position Specific Scoring Matrix
Zia-ur Rehman and Asifullah Khan
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00447]


Editorial:
[BSP/PPL/E-Pub/00448]


A Dialogue About Protein Crystallization and Phase Diagrams
Neer Asherie
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00449]


Randomness in Crystallization of Proteins from Staphylococcus Aureus
Shaomin Yan and Guang Wu
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00450]


Difficult Macromolecular Structures Determined Usingx-Ray Diffraction Techniques
Alejandra Hernández-Santoyo
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00451]


Predicting Protein Crystallizability and Nucleation
Nuria Sanchez-Puig, Claude Sauter, Bernard Lorber, Richard Giegé and Abel Moreno
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00452]


Bioactive Peptides From Marine Organisms: A Short Overview
Fernando Lazcano-Pérez, Sergio A. Román-González, Nuria Sánchez-Puig and Roberto Arreguín-Espinosa
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00453]


Enhanced Crystallizability By Protein Engineering Approaches: A General Overview
Alessia Ruggiero, Giovanni Smaldone, Flavia Squeglia and Rita Berisio
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00454]


Perspectives On High-Throughput Technologies Applied To Protein Crystallisation
Emmanuel Saridakis
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00455]


In Situ Observation of Elementary Growth Processes of Protein Crystals by Advanced Optical Microscopy
Gen Sazaki, Alexander Van Driessche, Guoliang Dai, Masashi Okada, Takuro Matsui, Fermín Otálora, Katsuo Tsukamoto and Kazuo Nakajima
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00456]


Synchrotron Radiation In Life Sciences
Vivian Stojanoff, Paul Northrup, Ruth Pietri and Zhong Zhong
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00457]


Practical Physics Behind Growing Crystals Of Biological Macromolecules
Nadine Candoni, Romain Grossier, Zoubida Hammadi, Roger Morin and Stéphane Veesler
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00458]


Effect Of The Compatible Solute Ectoine On The Stability Of The Membrane Proteins
Arpita Roychoudhury, Dieter Häussinger and Filipp Oesterhelt 
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00460]


Influence of Prolactin and Calcium Gluconate Concentration on Permeation and Intestinal Absorption of Ca(II) Ions
Florian Ryszka, Rimantas Klimas, Barbara Dolińska and Katarzyna Łopata
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00461]


Recombinant Production, Isotope Labeling and Purification of ENOD40B, a Plant Peptide Hormone
Young Kee Chae, Marco Tonneli and John L. Markley
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00462]


A Novel Antilithiatic Protein From Tribulus Terrestris Having Cytoprotective Potency
Anshu Aggarwal, Simran Tandon, Surinder Kumar Singla and Chanderdeep Tandon
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00463]


A novel polygalacturonase-inhibiting protein (pgip) from lathyrus sativus l. Seeds
Rachele Tamburino, Angela Chambery, Augusto Parente and Antimo Di Maro
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00464]


Thermal Inactivation And Conformational Lock Of Bovine Carbonic Anhydrase
L. Alaei, A.A.Moosavi-Movahedi, H. Hadi, A.A. Saboury, F. Ahmad and M. Amani
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00465]


Heterologous Production of Death Ligands’ and Death Receptors’ Extracellular Domains: Structural Features and Efficient Systems
Michiro Muraki
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00466]


Evaluating Quinacrine As A Potential Amyloid Imaging Compound: Studies On Hen Egg White Lysozyme As Model System
Manjeet Kumar, Nandini Sarkar and Vikash Kumar Dubey
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00467]


Water Refined Solution Structure of the Human Grb7-SH2 Domain in Complex with the erbB2 Receptor Peptide pY1139
Sally C. Pias, Dennis L. Johnson, David E. Smith and Barbara A. Lyons
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00468]


Analysis of the Proteome of Common Bean (Phaseolus vulgaris L.) Roots after Inoculation with Rhizobium etli
Afshin Salavati, Alireza Taleei, Ali Akbar Shahnejat Bushehri and Setsuko Komatsu
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00469]


Purification and Characterization of a Novel Anti-proliferative Lectin from Morus alba L. Leaves
Mundekkad Deepa and Sulochana Priya
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00470]


Kinetic and Structural Studies on the Interactions of Heparin and Proteins of Human Seminal Plasma using Surface Plasmon Resonance
Vijay Kumar, Vikash Kumar Yadav, Md. Imtaiyaz Hassan, Abhay Kumar Singh, Sharmistha Dey, Sarman Singh, Tej P.Singh and Savita Yadav
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00471]


Protein-Protein Networks Construction and Their Relevance Measurement Based on Multi-Epitope-Ligand-Kartographie and Gene Ontology Data of T-cell Surface Proteins for Polymyositis
Fang-Zhen Li and Feng Gao
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00472]


Saturation Transfer Difference NMR Studies of the Interaction of the Proteinkinase CK2 with Peptides
Christoph Räuber and Stefan Berger
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00473]


Cytokine Production Induced By Marine Algae Lectins In Balb/C Mice Splenocytes
Ticiana Monteiro Abreu, Luana Maria Castelo Melo Silva, Edfranck Sousa Oliveira Vanderlei, Cristiane Moutinho Lagos de Melo, Valéria Rêgo Alves Pereira and Norma Maria Barros Benevides
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00474]


Biophysical Analysis of the Transition of an All α-Helical Greek-Key Protein intoAmyloid Fibrils Composed of β-Sheet Structure
Jason C. Collins and Lesley H. Greene
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00475]


Targeting The Epidermal Growth Factor Receptor: Exploring The Potential Of Novel Inhibitor N-(3-Ethynylphenyl)-6, 7-Bis (2-Methoxyethoxy) Quinolin-4-Amine Using Docking And Molecular Dynamics Simulation
Shipra Gupta, Gauri Misra, Mohan Chandra Pant and Prahlad Kishore Seth
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00476]


Predicting Protein Solubility by the General Form of Chou’s Pseudo Amino Acid Composition: Approached from Chaos Game Representation and Fractal Dimension
Xiao-Hui Niu, Xue-Hai Hu, Feng Shi and Jing-Bo Xia
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00477]


Site-Directed Mutagenesis Study Of The Ile140 In Conserved Hydrophobic Core Of Bcl-Xl
Xin Zhang, Ying Tan, Rui Zhao, Bizhu Chu, Chunyan Tan and Yuyang Jiang
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00478]


Binding Of A Bcl-2 Family Inhibitor To Bovine Serum Albumin: Fluorescence Quenching And Molecular Docking Study
Rui Zhao, Yonghua Xie, Ying Tan, Chunyan Tan and Yuyang Jiang
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00479]


Prediction Of Human Genes’ Regulatory Functions Based On Protein-Protein Interaction Network
Peng Gao, Qing-Ping Wang, Lei Chen and Tao Huang
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00480]


A Novel Approach For Secretion Of Heterologous Proteins With Correct N-Terminal Processing By Using Α-Factor Pre Sequence In Pichia Pastoris
Lu Shen, Zhenyun Liu, Fenli Xie, Xuerui Zhang and Shuhua Tan
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00481]

Machine Learning Study Of Classifiers Trained With Biophysiochemical Properties Of Amino Acids To Predict Fibril Forming Peptide Motifs
Smitha Sunil Kumaran Nair, N. V. Subba Reddy and Hareesha K. S
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00482]


Peptidoglycan Hydrolase Enterolysin A Recognizes Lipoteichoic Acid Chains In The Cell Walls Of Sensitive Bacteria
Lenka Malinicova, Katarina Dubikova, Maria Piknova, Peter Pristas and Peter Javorsky
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00483]


Secreted Nucleobindin-2 Inhibits 3t3-L1 Adipocyte Differentiation
Yuko Tagaya, Aya Osaki, Atsuko Miura, Shuichi Okada, Kihachi Ohshima, Koshi Hashimoto, Masanobu Yamada, Tetsurou Satoh, Hiroyuki Shimizu and Masatomo Mori
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00484]


Dynamical Properties Of Steric Zipper Polymorphs Formed By A Iapp-Derived Peptide
Francesca Stanzione, Alfonso De Simone, Luciana Esposito and Luigi Vitagliano
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00485]


A Simple And Universal Method To Express Protein In Unfused Form
Tianwen WANG, Aitao LI, Xingyuan Mab, Guocheng DU and Jian CHEN
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00486]


Ph-Sensitive Self-Associations Of The N-Terminal Domain Of Nbce1-A Suggest A Compact Conformation Under Acidic Intracellular Conditions
Harindarpal S. Gill
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00487]


SOMEViz: a Web Service for Site of Metabolism Estimating and Visualizing
Qian-Cheng Shen, Ming-Yue Zheng, Jing Lu, Cheng Luo, Wei-Liang Zhu, Kai-Xian Chen, Xiao-Min Luo and Hua-Liang Jiang
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00488]


Epitope Mapping And Identification Of Amino Acids Critical For Rabbits Igg-Binding To Linear Epitopes On Buffalo Beta-Lactoglobulin
Li Xin, Chen Hongbing, Gao Jinyan, Liu Fahui and Wen Xuefang
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00489]


Predicting The Classification Of Transcription Factors By Incorporating Their Binding Site Properties Into A Novel Mode Of Chou’s Pseudo Amino Acid Composition
Liang-Yun Ren, Yu-Sen Zhang and Ivan Gutman
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00490]


Expression In Pichia Pastoris And Biological Activity Of Avian Β-Defensin 6 And Its Mutant Peptide Without Cysteines
Yanping Cao, Qingquan Ma, Anshan Shan and Na Dong
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00491]


Purification and Characterization of a Lectin of the Swartzieae Legume Taxa
Andreia Varmes Fernandes, Márcio Viana Ramos, Ilka Maria Vasconcelos, Ana Cristina Oliveira Monteiro Moreira, Frederico Bruno Moreno, Jose Odair Pereira and José Francisco de Carvalho Gonçalves
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00492]


A Method for Isolation of DNA-binding Proteins Based on Solubility of DNA-protein Complexes
Hua Yang, Huang Li, Li-qun Rao, Gui-you Long, Guo-ping Peng and Lu Jin
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00493]


L-Arginine Mediated Renaturation Enhances Yield Of Human, α6 Type Iv Collagen Non-Collagenous Domain From Bacterial Inclusion Bodies
Venugopal Gunda, Chandra Shekhar Boosani, Raj Kumar. Verma, Chittibabu Guda and Yakkanti Akul Sudhakar
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00494]


Hydropathic Self-Organized Criticality: A Magic Wand for Protein Physics
J. C. Phillips
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00495]


Proteome-Wide Inference of Human Endophilin 1-Binding Peptides
Gang Wu, Zeng-Li Zhang, Chun-Jiang Fu, Feng-Lin Lv and Fei-Fei Tian
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00496]


Characterization of the Interaction Between Endostatin Short Peptide and VEGF Receptor 3
Kyu-Yeon Han, Dimitri T. Azar,Abdellah Sabri, Hyun Lee, Sandeep Jain, Bao-Shiang Lee and Jin-Hong Chang
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00497]


Functional Analysis of Hybrid Peptide CAMA-Syn: Expression in Mammalian Cells and Antimicrobial Potential
Junlin Zhang, Sha Peng, Xiang Cheng and Huayan Wang
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00498]


Purification Of Pectin Methylesterase From Lycopersicon Esculentum And Its Application
Shashi kant and Reena Gupta
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00499]


Crystal Structure of a Flavin-dependent Thymidylate Synthase from Helicobacter pylori strain 26695
Xiaoli Zhang, Jinyong Zhang, Gang Guo, Xuhu Mao, Yonglin Hu and Quanming Zou
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00500]


Inactivation Kinetics Of β-N-Acetyl-D-Glucosaminidase From Green Crab (Scylla Serrata) By Guanidinium Chloride And The Relationship Between Its Enzyme Activity And Conformation
Ji-Ping Zhang, Bo Leng, Qian-Sheng Huang , Ya-Wen Yan, Xuan Liu, Qin Wang and Qing-Xi Chen
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00501]


Design, Synthesis, And Activity Evaluation Of A New 5-Fluorouracil Prodrug Containing An Asn-Gly-Arg(No2)Cooch3 Tripeptide
Yepeng Luan, Fanbo Jing, Jian Zhang, Mingming Zou, Xuejian Wang, Yuping Jia, , Ning Liu, Jiajia Mou and Wenfang Xu
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00502]


Editorial: Cell Processes in Bacterial Diseases
[BSP/PPL/E-Pub/00503]


Bacterial Lipopolysaccharides in Plant and Mammalian Innate Immunity
Cristina De Castro, Otto Holst, Rosa Lanzetta, Michelangelo Parrilli  and Antonio Molinaro
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00504]


Impact of Structural Domains of the Heparin Binding Hemagglutinin of Mycobacterium tuberculosis on Function
Giovanni Delogu, Giovanni Fadda and Michael J. Brennan
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00505]


Resuscitation-Promoting Factors (RPF): In Search of Inhibitors
Arseny S. Kaprelyants, Galina V. Mukamolova, Alessia Ruggiero, Vadim A. Makarov, Galina R. Demina, Margarita O. Shleeva, Vasilii D. Potapov and Pavel A. Shramko
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00506]


Structural And Dynamic Properties Of Incomplete Immunoglobulin-Like Fold Domains
Rita Berisio, Luciano Ciccarelli , Flavia Squeglia, Alfonso De Simone and Luigi Vitagliano
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00507]


Eight Stranded β-barrel and Related Outer Membrane Proteins: Role in Bacterial Pathogenesis
Siobhán McClean
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00508]


Isolation and Identification of Novel Neutrophil-Activating Cryptides Hidden in Mitochondrial Cytochrome c
Yoshinori Hokari, Tetsuo Seki, Hiroko Nakano, Yuko Matsuo, Akiyoshi Fukamizu, Eisuke Munekata, Yoshiaki Kiso and Hidehito Mukai
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00522]


Local Flexibility Facilitates Oxidization of Buried Methionine Residues
Kuiran Xu, Vladimir N. Uversky and Bin Xue
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00523]


Detection of Antibodies against Synthetic Peptides Mimicking Ureases Fragments in Sera of Rheumatoid Arthritis Patients
Iwona Konieczna, Marek Kwinkowski, Beata Kolesinska, Zbigniew Kaminski, Justyna Fraczyk, Paulina Zarnowiec and Wieslaw Kaca
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00524]


Biochemical property and in vivo efficacies of novel Val/Arg-rich antimicrobial peptide
Qing-Quan Ma, Na Dong, An-Shan Shan , Liang Wang, Wan-Ning Hu and Wen-Yu Sun
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00525]


Novel design of short antimicrobial peptides derived from the bactericidal domain of avian ß-defensin-4
Na Dong, Qing-Quan Ma, An-Shan Shan , Yin-Feng Lv, Wanning Hu, Yao Gu and Yu-Zhi Li
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00526]


Crystal structure of the Tum1 protein from the yeast Saccharomyces cerevisiae
Rui Qiu, Fengbin Wang, Meiruo Liu, Tiantian Lou and Chaoneng Ji
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00527]


Biochemical characterization of thymidine monophosphate kinase from white spot syndrome virus: A functional domain from the viral ORF454
Eduardo Guevara-Hernandez, Karina D. Garcia-Orozco and Rogerio R. Sotelo-Mundo
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00528]


Aqueous Microwave-Assisted Solid-Phase Peptide Synthesis Using Fmoc Strategy: In-Water Synthesis of “Difficult Sequences”
Keiko Hojo, Hideki Ichikawa, Asaki Hara, Mare Onishi, Koichi Kawasaki and Yoshinobu Fukumori
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00529]


Characterization of Glycine Substitution Mutations within the Putative NAD+-binding Site of Bacillus licheniformis Aldehyde Dehydrogenase
Yen-Chung Lee, Den-Tai Lin, Hsiang-Ling Chen, Huei-Fen Lo, Hui-Yu Hu, Nai-Wan Hsiao and Long-Liu Lin
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00530]


Principles, Challenges and Advances in ab initio Protein Structure Prediction
Arunachalam Jothi
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00531]


SVM prediction of ligand-binding sites in bacterial lipoproteins employing shape and physio-chemical descriptors
Kiran Kadam, Prashant Prabhakar and V. K. Jayaraman
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00532]



Abstracts



An Rgd-modified Endostatin Peptide Expressed at e. Coli shows Anti-tumor Activity in vivo
Hai Tao Zhang,  Hui Cheng Li,   Zheng Wu Li,  ChangHong Guo,  Yu Jun Chen
 [FULL-TEXT INQUIRY] [PMID: 21675950 PubMed - indexed for MEDLINE] [BSP/PPL/E-Pub/00349]

Tumor vasculatures express high levels of αVβ3/αVβ5 and α5β1 integrins. Peptide containing the RGD (Arg-Gly-Asp) sequence, which is present in ligands of integrins, is effective in targeting therapeutic reagents to tumor vascular endothelium. In this study, we investigated whether the biological activity of endostatin 27 peptides can be enhanced by the addition of an integrin targeting sequence. RGDRGD and GGGRGD sequence were added to the carboxyl terminus of endostatin 27 and 25 peptides, respectively. Modification of endostatin 27 peptides with the RGD motif showed specific and increased binding to endothelial cells and the increased binding is consistent with improved antiangiogenic property. RGD-modified endostatin 27 peptides was more effective than human endostatin and endostatin 27 peptides in inhibiting liver cancer growth in athymic mice. These finding indicates that addition of a vascular targeting sequence can enhance the biological activity of an antiangiogenic peptides molecule.
[Back to top]


Predicting Viral Protein Subcellular Localization with Chou’s Pseudo Amino Acid Composition and Imbalance-Weighted Multi-Label K-Nearest Neighbor Algorithm
Jun-Zhe Cao andHong Gu
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00431]

Machine learning is a kind of reliable technology for automated subcellular localization of viral proteins within a host cell or virus-infected cell. One challenge is that the viral protein samples are not only with multiple location sites, but also class-imbalanced. The imbalanced dataset often decreases the prediction performance. In order to accomplish this challenge, this paper proposes a novel approach named imbalance-weighted multi-label K-nearest neighbor to predict viral protein subcellular location with multiple sites. The experimental results by jackknife test indicate that the presented algorithm achieves a better performance than the existing methods and has great potentials in protein science.
[Back to top]


Incorporating Secondary Features Into The General Form Of Chou’s Pseaac For Predicting Protein Structural Class
Bo Liao,Qilin Xiang and Dachao Li
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00432]

Protein structure information is very useful for the confirmation of protein function. The protein structural class can provide information for protein 3D structure analysis, causing the conformation of the protein overall folding type plays a significant part in molecular biology. In this paper, we focus on the prediction of protein structural class which was based on new feature representation. We extract features from the Chou-Fasman parameter, amino acid compositions, amino acids hydrophobicity features, polarity information and pair-coupled amino acid composition. The prediction result by the SVM classifier shows that our method is better than some others.
[Back to top]


Epitope Mapping And Identification Of Amino Acids Critical For Rabbits Igg-Binding To Linear Epitopes On Buffalo Beta-Lactoglobulin
Li Xin, Chen Hongbing, Gao Jinyan, Liu Fahui and Wen Xuefang
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00446]

Buffalo milk safety was highlighted with the increase in dietary consumption, and a little information is available on buffalo milk allergy except for cross-reactivity between buffalo and cow milk. In this work, linear epitopes and critical amino acids of buffalo β-lactoglobulin were defined by 4 rabbit’s sera using SPOTTM peptide arrays approach based on the defined mimotope. The eight epitopes on buffalo β-lactoglobulin were located in the position of A6(21-30), A7(AA25-34), A8 (29-38), B4 (73-82), B5(77-86), C(87-96), F4(134-143) and F8(150-159), respectively. Among them, four epitopes (A7, A8, F4 and F8) were described as the most major epitopes and peptide (A6, B4, B5 and C) as the second major epitopes. Following single AA substitutions (Alanine or Glycine) at each position of the major epitopes, 2，3，2，3，5 and 3 of critical amino acids were identified on epitopes of A6, A8, B5, C , F4 and F8, respectively, which vary in distribution among the epitopes, such as in C terminal or N terminal and in continuous or discontinuous forms, characteristics including hydrophobicity, polar and charge, and existed frequency.
[Back to top]


Identify Gpcrs And Their Types With Chou’s Pseudo Amino Acid Composition: An Approach From Multi-Scale Energy Representation And Position Specific Scoring Matrix
Zia-ur Rehman and Asifullah Khan
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00447]

G-protein coupled receptor (GPCR) is a membrane protein family which serves as an interface between cell and the outside world. They are involved in various physiological processes and are the targets of more than 50% of the marketed drugs. The function of GPCRs can be known by conducting biological experiments. However, with the rapid increase of GPCR sequences entering into databanks, it is very time-consuming and expensive to determine their functions based only on experimental techniques. Hence, the computational prediction of GPCRs is very much demanding for both pharmaceutical and basic research. Feature extraction of GPCRs in the proposed research was performed via the following three approaches: (i) pseudo amino acid composition, (ii) wavelet based multi-scale energy, and (iii) evolutionary information derived through the position specific scoring matrices. For classification purpose, a majority voting based ensemble method was used, and their weights were optimized using genetic algorithm. Four classifiers were used in the ensemble system; they were Nearest Neighbor, Probabilistic Neural Network, Support Vector Machine and Grey Incidence Degree. The performance of the proposed method was evaluated by the jackknife tests for a number of datasets. First, the individual performances of classifiers were assessed for each dataset. After that, the performance for each dataset was improved by using weighted ensemble classification. The weights in the ensemble classifier were optimized by using various runs of Genetic Algorithm. We have compared our method with various other methods, and the outcomes indicated that the proposed method would be quite useful for GPCRs classification.
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Editorial:
[BSP/PPL/E-Pub/00448]

This “Hot Topic” thematic issue is the result of nearly20 years of the Guest Editor'sexpertise on protein crystallization and protein crystal growth methods. In this issue we deal with most of the basic problems on protein crystallization that have to be faced by PhD students in research laboratories, as well as by postdoctoral fellows and researchers. The recent advances and points of view in over-expression methods from molecular biology and protein engineering have been properly revised. The analyses of the main problems in protein crystallization, themethods to grow crystals, and thestrategies to analyze them by dynamic light scattering (even a scientific poem to explain the theory is included), and X-ray diffraction methodsaredevoted to difficult targets in the majority of the cases. The scope of this issue is in generalconcentrated on theoretical and experimental approaches in order to deal with the 3D structure of biological macromolecules, starting from the protein over-expression going through nucleation, predicting protein crystallization, protein crystallization methods, investigations on condensed phases and phase diagrams, to obtain the 3D structure of a variety of proteins and even marine peptides, which are difficult to crystallize. We have also introduced a substantial revision of physical and chemical strategies that we hope will help to understand the process of protein crystallization as well as the obtaining of high-quality protein single crystals for high-resolution X-ray Crystallography.

This is the first time that a group of experts in each topic of the reviews, letters or research papers include frontier ideas to deal with protein crystallization for novel and difficult biological targets, exactly for those recalcitrant to be crystallized. The basics of each topic are carefully revised and most of the reviews are connected as a trend-line to guide the readers to understand the fundamentals of protein crystallization process applied to the crystallization of difficult targets and how to sort it out. We hope that all this effort will help and inspire new generations of scientists to understand that this knowledge is not just for solus scientist, but formultidisciplinary scientists especially whentrying to solve all multifactorial problems in protein crystallization. This issue is devoted to thosewho are interested in the way of doing Science in the New Millennium.

Finally, I would like to acknowledge the help and contributions of all my colleagues and friends that have made a great effort to complete these reviews on time, and forme to be able toput this volume together. I would also like to acknowledge the effort andpatience from the Editor-in-Chief of Protein and Peptide Letters, and the help of the Editorial Managing team whiledealing with authors and reviewers. WhenI was invited two years ago to beguest editor of another special issue, during my sabbatical in England, I realized that a second issue with extra information would be necessary, and that thisissue should contain reviews about new and classic theories, experimental approaches,and recent contributions (based in Bioinformatics and Neural Networks) for the crystallization of difficult targets. I also realized that in order to do this I would need the contributions of the rest of my colleagues that for reason of time and number could not have been invited to that first issue while I was in England, as they are also leading scientistsin the fieldsof Nucleation Phenomena, Physics of Crystal Growth, Dynamic Light Scattering, Protein Crystallography, Advanced Optical Microscopy, High-throughput Technologies, Molecular Biology, Protein Engineering, Marine Peptide's Biochemistryas well as in Bioinformatics. This is the result and The Master Pieceofall of them together.
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A Dialogue About Protein Crystallization and Phase Diagrams
Neer Asherie
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00449]

A lighthearted researcher and a disheartened student discuss the challenges of protein crystallization and how phase diagrams can be used to address these challenges. The student feels a little better afterwards, but many proteins remain uncrystallized.



Randomness in Crystallization of Proteins from Staphylococcus Aureus
Shaomin Yan and Guang Wu
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00450]

Of many factors affecting protein crystallization, randomness in proteins has been given less attention although highly structured proteins would be at low entropy state. The factors, which impact on protein crystallization, are almost exclusively related to non-random amino acid properties such as physiochemical properties of amino acids. In this study, we used logistic regression and neural network to model the success rate of crystallization of 420 proteins from Staphylococcus aureus with each of non-random and random amino acid properties in order to determine whether randomness in a protein plays a role in the crystallization process. The results show that randomness is indeed involved in the crystallization process, and this rationale would enrich our knowledge on crystallization process and enhance our ability to crystallize more important proteins.
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Difficult Macromolecular Structures Determined Usingx-Ray Diffraction Techniques
Alejandra Hernández-Santoyo
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00451]

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexesand assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this techniquecomplemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.
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Predicting Protein Crystallizability and Nucleation
Nuria Sanchez-Puig, Claude Sauter, Bernard Lorber, Richard Giegé and Abel Moreno
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00452]

The outcome of protein crystallization attempts is often uncertain due to inherent features of the protein or to the crystallization process that are not fully under control of the experimentalist. The aim of this contribution is to propose user-friendly tools that can increase the success rate of a protein crytallization project. It provides different bioinformatic approaches to predict the crystallization feasibility (before any crystallization attempts are undertaken) and a novel approach to assess the nucleation process of a given protein. Practical examples illustrate these two points.
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Bioactive Peptides From Marine Organisms: A Short Overview
Fernando Lazcano-Pérez, Sergio A. Román-González, Nuria Sánchez-Puig and Roberto Arreguín-Espinosa
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00453]

Marine organisms are an immense source of new biologically active compounds. These compounds are unique because the aqueous environment requires a high demand of specific and potent bioactive molecules. Diverse peptides with a wide range of biological activities have been discovered, including antimicrobial, antitumoral, and antiviral activities and toxins amongst others. These proteins have been isolated from different phyla such as Porifera, Cnidaria, Nemertina, Crustacea, Mollusca, Echinodermata and Craniata. Purification techniques used to isolate these peptides include classical chromatographic methods such as gel filtration, ionic exchange and reverse-phase HPLC. Multiple in vivo and in vitro bioassays are coupled to the purification process to search for the biological activity of interest. The growing interest to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs now in clinical trials. This review presents examples of interesting peptides obtained from different marine organisms that have medical relevance. It also presents some of the common methods used to isolate and characterize them.
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Enhanced Crystallizability By Protein Engineering Approaches: A General Overview
Alessia Ruggiero, Giovanni Smaldone, Flavia Squeglia and Rita Berisio
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00454]

The limiting step in macromolecular crystallography is the preparation protein crystals suitable for X-ray diffraction studies. A strong prerequisite for the success of crystallization experiments is the ability to produce monodisperse and properly folded protein samples. Since the production of most protein is usually achieved using recombinant methods, it has become possible to engineer target proteins with increased propensities to form well diffracting crystals. Recent advances in bioinformatics, which takes advantage from an enhanced information in the protein databases, are of enormous help for the design of modified proteins. Based on bioinformatics analyses, the reduction of the structural complexity of proteins or their site-specific mutagenesis has proven to have a dramatic impact on both the yield of heterologous protein expression and its crystallizability.

Therefore, protein engineering represents a valid tool which supports the classical crystallization screenings with a more rational approach. This review describes key methods of protein-engineering and provides a number of examples of their successful use in crystallization.
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Perspectives On High-Throughput Technologies Applied To Protein Crystallisation
Emmanuel Saridakis
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00455]

High-throughput crystallisation requires the rapid and accurate dispensing of protein and precipitating agent solutions at nanovolumes, but does not end there. The choice of the initial screens is very important, especially with respect to the availability of protein material. Data from previous crystallisation experiments that are scattered in the literature and only partially available in databases have to be analysed in efficient ways that will maximise their utility for designing new screens. A larger portion of crystallisation parameter space should be made accessible to screening, through the use of nucleants and seeding. Observation, assessment and scaling up of the crystallisation trials should be efficiently performed and, finally yet importantly, optimisation of conditions must also be adapted to the high-throughput environment. The above requirements are briefly addressed in the following paper.
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In Situ Observation of Elementary Growth Processes of Protein Crystals by Advanced Optical Microscopy
Gen Sazaki, Alexander Van Driessche, Guoliang Dai, Masashi Okada, Takuro Matsui, Fermín Otálora, Katsuo Tsukamoto and Kazuo Nakajima
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00456]

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.
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Synchrotron Radiation In Life Sciences
Vivian Stojanoff, Paul Northrup, Ruth Pietri and Zhong Zhong
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00457]

Synchrotron Radiation (SR) presents itself as a “play-ground” with a large range of methods and techniques suitable to unveil the mysteries of life. Here we attempt to present a few of these methods that complement those employed in the home laboratory. SR diffraction, spectroscopy and imaging methods relevant to the atomic structure determination and characterization of the properties and function of chemical compounds and macromolecules of biological relevance, are introduced.
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Practical Physics Behind Growing Crystals Of Biological Macromolecules
Nadine Candoni, Romain Grossier, Zoubida Hammadi, Roger Morin and Stéphane Veesler
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00458]

The aim of this review is to provide biocrystallographers who intend to tackle protein-crystallization with theory and practical examples. Crystallization involves two separate processes, nucleation and growth, which are rarely completely unconnected. Here we give theoretical background and concrete examples illustrating protein crystallization. We describe the nucleation of a new phase, solid or liquid, and the growth and transformation of existing crystals obtained by primary or secondary nucleation or by seeding. Above all, we believe that a thorough knowledge of the phase diagram is vital to the selection of starting position and path for any crystallization experiment.
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Effect Of The Compatible Solute Ectoine On The Stability Of The Membrane Proteins
Arpita Roychoudhury, Dieter Häussinger and Filipp Oesterhelt 
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00460]

Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.
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Influence of Prolactin and Calcium Gluconate Concentration on Permeation and Intestinal Absorption of Ca(II) Ions
Florian Ryszka, Rimantas Klimas, Barbara Dolinska and Katarzyna Lopata
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00461]

The in vitro permeation and absorption of calcium ions across the small intestine were measured at different concentrations of calcium gluconate solutions (1.0, 10.0 and 20.0 mM) with or without prolactin. The calcium ions permeated through the small intestine from a donor environment to an acceptor environment that mimicked the conditions in the stomach to ileum segment of the digestive tract. The permeation and absorption of calcium were directly dependent on the calcium concentration of the solutions. At 10 and 20 mM permeation was significantly higher than that at 1.0 mM (p<0.05). In the presence of prolactin both permeation and absorption increase considerably. At the lowest concentration (1.0 mM) simulating calcium deficiency, there was compensation by the small intestine, suggesting that such deficiency stimulates its mobilization from intestinal tissue. Prolactin enhances the calcium mobilization process even at sufficient calcium intakes. It is suggested that prolactin takes part in regulation of calcium homeostasis in the organism.
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Recombinant Production, Isotope Labeling and Purification of ENOD40B, a Plant Peptide Hormone
Young Kee Chae, Marco Tonneli and John L. Markley
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00462]

The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDI-TOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.
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A Novel Antilithiatic Protein From Tribulus Terrestris Having Cytoprotective Potency
Anshu Aggarwal, Simran Tandon, Surinder Kumar Singla and Chanderdeep Tandon
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00463]

Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.
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A novel polygalacturonase-inhibiting protein (pgip) from lathyrus sativus l. Seeds
Rachele Tamburino, Angela Chambery, Augusto Parente and Antimo Di Maro
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00464]

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant proteins bound to the plant cell wall containing leucine-rich repeats (LRR). They play an important role in plant defence being able to inhibit fungal endopolygalacturonases (EPGs), the first enzymes secreted by phytopathogenic fungi during plant infection. In the present work, a novel PGIP (LsPGIP) has been isolated from Lathyrus sativus seeds. LsPGIP exhibited an inhibitory activity towards EPGs from Aspergillus niger and Rhizopus spp. A pI value of 8.3 and a molecular mass of 40 kDa were determined for the purified inhibitor. Furthermore, N-terminal sequence up to residue 20 revealed that LsPGIPexhibit a high percentage of identity with PGIP fromActinidia deliciosa. A secondary structure similar to those of other polygalacturonase inhibitors was also inferred form circular dichroism data.
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Thermal Inactivation and Conformational lock of Bovine carbonic Anhydrase
L. Alaei, A.A.Moosavi-Movahedi, H. Hadi, A.A. Saboury, F. Ahmad and M. Amani
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00465]

The kinetics of thermal inactivation of bovine carbonic anhydrase (BCA) was studied in a 50 mM Tris-HCl buffer, pH 7.8 using p-nitrophenyl acetate as substrate in absorbance of 400 nm by UV-VIS spectrophotometry. The number of conformational locks and inter-subunit amino acid residues of BCA were obtained by thermal inactivation analysis. The cleavage bonds between dimers of BCA during thermal dissociation and type of interactions between specific amino acid residues were also detected. The thermal inactivation curves were plotted in temperatures ranging between 40-70°C. It was shown several phases for inactivation of BCA at 65°C. Analyses of the curves were done by the conformational lock theory. The subunits are dissociated and several intermediates appear during inactivation through increasing the temperature in comparison with native state. Dynamic light scattering measurements was done to study the changes in hydrodynamic radius during thermal inactivation. Three distinct zones were shown in DLS data. Biochemical computation using ligplot is performed to find the inter-subunit amino acid residues for BCA.
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Heterologous Production of Death Ligands’ and Death Receptors’ Extracellular Domains: Structural Features and Efficient Systems
Michiro Muraki
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00466]

The extracellular domains of death ligands and those of death receptors are closely related to many serious human diseases through the initiation of apoptosis.  Recombinant production of the extracellular domains has been investigated due to demand for a large amount of purified samples, which are a prerequisite for their biochemical characterization and constitute the fundamentals of medical applications.  This review focuses on the recombinant production of extracellular domains of the major members of death ligand and death receptor families using non-mammalian expression systems with an emphasis on Fas ligand and Fas receptor.  In contrast to the efficient production of the functional extracellular domains of TRAIL, TNFα and LTα by intracellular expression systems using Escherichia coli or Pichia pastoris, that of Fas ligand requires the secretory expression systems using P. pastoris or Dictyostelium discoideum, and the productivity in P. pastoris was largely dependent on tag sequence, potential N-glycosylation site and expressed protein region.  On the other hand, the exploitation of insect cell systems is generally useful for the preparation of functional extracellular domains of death receptors containing many disulfide bridges in the absence of extended secondary structure, and a Bombyx mori larvae secretion system presented a superior productivity for human Fas receptor extracellular domain.  Based on the results obtained so far, further efforts should be devoted to the artificial control of death ligand – death receptor interactions in order to make a contribution to medicine, represented by the development of novel biopharmaceuticals.
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Evaluating Quinacrine As A Potential Amyloid Imaging Compound: Studies On Hen Egg White Lysozyme As Model System
Manjeet Kumar, Nandini Sarkar and Vikash Kumar Dubey
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00467]

Amyloid plaque is associated with several neuronal and non-neuronal degenerative diseases. More than twenty human proteins can fold abnormally to form pathological deposits like amyloid plaque. Strategies for treating such diseases include therapies designed to decrease protein plaque formation or its complete clearance, but monitoring/clinical trials of these treatments are limited by the lack of effective methods to monitor amyloid deposits in the organs/tissues of living patients. The current study shows binding and staining ability of quinacrine to protein amyloid deposits, using Hen Egg White Lysozyme (HEWL) as model system and characterization of its binding interaction with HEWL, employing several biophysical techniques. Since quinacrine can pass the blood brain barrier, the current report suggests potential application of quinacrine for antemortem diagnostic of amyloid.
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Water Refined Solution Structure of the Human Grb7-SH2 Domain in Complex with the erbB2 Receptor Peptide pY1139
Sally C. Pias, Dennis L. Johnson, David E. Smith and Barbara A. Lyons
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00468]

We report a refinement in implicit water of the previously published solution structure of the Grb7-SH2 domain bound to the erbB2 receptor peptide pY1139. Structure quality measures indicate substantial improvement, with residues in the most favored regions of the Ramachandran plot increasing by 14 % and with WHAT IF statistics (Vriend, G. J Mol Graph,1990, 8(1), 52-56) falling closer to expected values for well-refined structures.
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Analysis of the Proteome of Common Bean (Phaseolus vulgaris L.) Roots After Inoculation With Rhizobium etli
Afshin Salavati, Alireza Taleei, Ali Akbar Shahnejat Bushehri and Setsuko Komatsu
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00469]

Proteomics techniques were used to identify the underlying mechanism of the early stage of symbiosis between the common bean (Phaseolus vulgaris L.) and bacteria. Proteins from roots of common beans inoculated with bacteria were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. From 483 protein spots, 29 plant and 3 bacterial proteins involved in the early stage of symbiosis were identified. Of the 29 plant proteins, the expression of 19 was upregulated and the expression of 10 was downregulated. Upregulated proteins included those involved in protein destination/storage, energy production, and protein synthesis; whereas the downregulated proteins included those involved in metabolism. Many upregulated proteins involved in protein destination/storage were chaperonins and proteasome subunits. These results suggest that defense mechanisms associated with induction of chaperonins and protein degradation regulated by proteasomes occur during the early stage of symbiosis between the common bean and bacteria.
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Purification and Characterization of a Novel Anti-proliferative Lectin from Morus alba L. Leaves
Mundekkad Deepa and Sulochana Priya
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00470]

A novel anti-proliferative lectin was purified from Morus alba L. (Mulberry) leaves by a two step chromatographic procedure namely, immobilized metal ion affinity chromatography (IMAC) and convective interaction media (CIM) based anion exchange chromatography. The purified mulberry leaf lectin (MLL) was specific to galactose, galactosamine and N-acetyl galactosamine (GalNAc). MLL was homogenous with a molecular weight of ~56kDa in silver stained SDS-PAGE. The lectin showed RBC agglutination activity up to 40°C and was independent of pH above pH 6. Haemagglutination activity of purified MLL was not dependent on any metal ions. However, with high concentration of trivalent metal ions, Fe3+ and Al3+ and the divalent metal ion Fe2+,  a three fold increase in agglutination activity was observed. The purified MLL showed an anti-proliferative activity towards human breast cancer cells (MCF-7) and colon cancer cells (HCT-15) with a higher potency towards MCF-7 cells. This is the first report on the anti-proliferative activity of a GalNAc specific lectin from M. alba.
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Kinetic and Structural Studies on the Interactions of Heparin and Proteins of Human Seminal Plasma using Surface Plasmon Resonance
Vijay Kumar, Vikash Kumar Yadav, Md. Imtaiyaz Hassan, Abhay Kumar Singh, Sharmistha Dey, Sarman Singh, Tej P.Singh and Savita Yadav
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00471]

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dose-dependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.
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Protein-Protein Networks Construction and Their Relevance Measurement Based on Multi-Epitope-Ligand-Kartographie and Gene Ontology Data of T-cell Surface Proteins for Polymyositis
Fang-Zhen Li and Feng Gao
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00472]

Polymyositis is an inflammatory myopathy characterized by muscle invasion of T-cells penetrating the basal lamina and displacing the plasma membrane of normal muscle fibers. In order to understand the different adhesive mechanisms at the T-cell surface, Schubert randomly selected 19 proteins expressed at the T-cell surface and studied them using MELK technique [4], among which 15 proteins are picked up for further study by us. Two types of functional similarity networks are constructed for these proteins. The first type is MELK similarity network, which is constructed based on their MELK data by using the McNemar’s test [24]. The second type is GO similarity network, which is constructed based on their GO annotation data by using the RSS method to measuring functional similarity. Then the subset surprisology theory is employed to measure the degree of similarity between two networks. Our computing results show that these two types of networks are high related. This conclusion added new values on MELK technique and expanded its applications greatly.
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Saturation Transfer Difference NMR Studies of the Interaction of the Proteinkinase CK2 with Peptides
Christoph Räuber and Stefan Berger
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00473]

Saturation Transfer Difference NMR (STD NMR) is used for the detection of the binding constant of the decapeptide RRRDDDSDDD with CK2α, the catalytic subunit of the proteinkinase CK2. For this work a valid irradiation frequency of the CK2α had to be found ensuring that no peptide resonance is affected by the irradiation. This is the principle problem for investigations of protein peptide interactions by STD NMR due to the similarity of protein and peptide resonances. It is shown that by careful selection of the irradiation point a KD value averaging to 1 mM can be found. In addition, preferred binding sites of the peptide are detected and it is shown that the side chains of serine and aspartate are closest to the protein surface.
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Cytokine Production Induced By Marine Algae Lectins In Balb/C Mice Splenocytes
Ticiana Monteiro Abreu, Luana Maria Castelo Melo Silva, Edfranck Sousa Oliveira Vanderlei, Cristiane Moutinho Lagos de Melo, Valéria Rêgo Alves Pereira and Norma Maria Barros Benevides
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00474]

Marine algae can serve as sources of bioactive compounds and currently have been shown their potential biological and pharmaceutical applications. Marine algae lectins have been shown to be effective at controlling inflammatory processes. This work aimed to analyze the immunostimulatory properties of lectins from the marine algae Solieria filiformis (SfL), Pterocladiella capillacea (PcL) and Caulerpa cupressoides (CcL). This analysis was performed on BALB/c mouse splenocytes by measuring cytokine and nitric oxide production and cellular damage using tests of cytotoxicity and cell viability. These lectins were not cytotoxic (1-100 μg/mL), and were not able to induce IFN-γ and IL-2 production. IL-10 production was induced at high levels by all lectins tested. Treatment with SfL induced IL-6 production at higher levels at all experimental times, whereas treatment with PcL and CcL induced higher levels only in 24 and 72 h. Treatment with SfL did not result in nitrite oxide production, whereas treatment with PcL or CcL was able to induce nitrite release at high levels (after 24, 48 and 72 h). Lesser cellular damage (5%) was observed in splenocytes treated with these lectins (10 μg/mL). Thus, the lectins from these algae were not cytotoxic, promoted increased in cell viability and induced Th2 immune responses in mouse splenocytes, indicating that they have anti-inflammatory effects.
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Biophysical Analysis of the Transition of an All α-Helical Greek-Key Protein intoAmyloid Fibrils Composed of β-Sheet Structure
Jason C. Collins and Lesley H. Greene
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00475]

Amyloidosis resulting from the deposition of aggregated protein has been linked to many debilitating degenerative diseaseswhich include most notably Alzheimer’s and Parkinson’s. The tendency for a protein to alternatively form highly ordered amyloid fibrils is dependent on many biological factors. Mutations, temperature, concentration, translational motion and pH play a pivotal role in inducing fibril aggregate assembly in vitro. The key feature appears to be the need to destabilize the native state structure as a required first step. In this paper we report on the detailed conversion of the death domain ofthe human Fas-associated death domain, an all α-helical protein with a Greek-key topology, into an all β-sheet amyloid fibril, using a comprehensive range of spectroscopic techniques that provide insight into this process. This transition from α-helical to β-sheet seems to require destabilization but not complete loss of the secondary structure to explore alternative conformations. This is a fascinating transition that supports the hypothesis that all proteins have the innate ability to form a fibril-like structure. Thus the primary structure can encode two alternative three-dimensional structures: the native, functional state and the β-amyloid state. The Fas-associated death domain does not appear tonaturally form amyloid fibrils in vivo. Our results clearly indicate that proteins evolved to avoid amyloid fibril formation because we find that the conditions required for formation in our model system are very specific and far from physiological.
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Targeting The Epidermal Growth Factor Receptor: Exploring The Potential Of Novel Inhibitor N-(3-Ethynylphenyl)-6, 7-Bis (2-Methoxyethoxy) Quinolin-4-Amine Using Docking And Molecular Dynamics Simulation
Shipra Gupta, Gauri Misra, Mohan Chandra Pant and Prahlad Kishore Seth
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00476]

Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer related death. The epidermal growth factor receptor (EGFR) pathway is over expressed in HNSCC. EGFR regulates the HNSCC by inducing signalling events responsible for regulating key tumorigenic processes such as proliferation, inhibition of apoptosis, cell adhesion/ motility, growth and survival.

Present study evaluates the potential of N-(3-Ethynylphenyl)-6, 7-bis (2-methoxyethoxy) quinolin-4-amine as a new inhibitor for EGFR. We have explored the binding and inhibitory potential of the compound using molecular docking, structural interactions fingerprinting and molecular dynamics studies. The inhibitor exhibits extensive interactions with the EGFR catalytic site in the form of hydrogen bonds, pi-pi bond and salt bridges. It shows high specificity and binding affinity towards the protein.

The compound can further be explored for its potential to serve in the diagnosis and treatment of HNSCC. The quantitative prediction provides a scope for future experimental testing, facilitating the understanding of the crosstalks between signalling pathways.
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Predicting Protein Solubility by the General Form of Chou’s Pseudo Amino Acid Composition: Approached from Chaos Game Representation and Fractal Dimension
Xiao-Hui Niu, Xue-Hai Hu, Feng Shi and Jing-Bo Xia
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00477]

Obtaining soluble proteins in sufficient concentrations is a major obstacle in various experimental studies. How to predict the propensity of targets in large-scale proteomics projects to be soluble is a significant but not fairly resolved scientific problem. Chaos game representation (CGR) can investigate the patterns hiding in protein sequences, and can visually reveal previously unknown structure. Fractal dimensions are good tools to measure sizes of complex, highly irregular geometric objects. In this paper, we convert each protein sequence into a high-dimensional vector by CGR algorithm and fractal dimension, and then predict protein solubility by these fractal features together with Chou’s pseudo amino acid composition features and support vector machine (SVM). We extract and study six groups of features computed directly from the primary sequence, and each group is evaluated by the 10-fold cross-validation test. As the results of comparisons, the group of 445-dimensional vector gets the best results, the average accuracy is 0.8741 and average MCC is 0.7358. The resulting predictor is also compared with existing methods and shows significant improvement.
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Site-Directed Mutagenesis Study Of The Ile140 In Conserved Hydrophobic Core Of Bcl-Xl
Xin Zhang, Ying Tan, Rui Zhao, Bizhu Chu, Chunyan Tan and Yuyang Jiang
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00478]

The hydrophobic core in Bcl-xL composed of Trp137, Ile140, Trp181, Ile182, Trp188 and Phe191 is highly conserved and essential for protein folding, protein stability and binding affinity with BH3-peptide.  9 mutants of Ile140 residue were constructed and characterized in order to get better understanding of the effect of the hydrophobic core.  Binding assay demonstrated that binding affinities between 4 charged mutants and BH3-peptide were significantly weakened or lost, suggesting that the integrity of the hydrophobic core has close relationship with binding.  The CD spectroscopy results indicated that disruption of the hydrophobic core may affect local conformation within the protein and result in intrinsic inactivity.  Further chemical-induced protein folding results on these 4 mutants revealed that the conserved hydrophobic core is also important for the protein stability.
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Binding Of A Bcl-2 Family Inhibitor To Bovine Serum Albumin: Fluorescence Quenching And Molecular Docking Study
Rui Zhao, Yonghua Xie, Ying Tan, Chunyan Tan and Yuyang Jiang
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00479]

Both fluorescence spectroscopic and molecular docking methods were used to investigate the interaction between bovine serum albumin (BSA) and a known Bcl-xl/Bcl-2 inhibitor HA 14-1.  Based on the spectral overlap between the emission of BSA and absorption of HA 14-1, Förster energy transfer was proposed to be the possible quenching mechanism.  The Stern-Volmer constants are 2.49 × 104, 2.04× 104 and 0.90 × 104 M-1 at 293, 303 and 318 K, respectively, indicating that a static quenching process dominates.  Thermodynamic parameters were further obtained.  The derived negative ΔH (-27.51 kJ mol-1) and ΔS (-11.11 J mol-1K-1) values suggest hydrogen bond interaction and van der Waals force are the main binding force. The docking study was performed on BSA model.  According to the docking score and the number of hydrogen bonds, the potential binding site for HA 14-1 is proposed to be the site IIA, also known as drug site 1.
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Prediction Of Human Genes’ Regulatory Functions Based On Protein-Protein Interaction Network
Peng Gao, Qing-Ping Wang, Lei Chen and Tao Huang
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00480]

In systems biology, regulatory pathway is one of the most important research areas. However, regulatory pathway is so complicated that we still poorly understand this system. On the other hand, with rapid accumulated information on different organisms, it becomes more and more possible to in-depth investigate regulatory pathway. To understand regulatory pathway well, figuring out the components of each pathway is the most important step. In this study, a network-based method was proposed to classify human genes into corresponding pathways. The information of protein-protein interactions retrieved from STRING was used to construct a network and jackknife test was employed to evaluate the method. As a result, the first order prediction accuracy was 87.91%, indicating that interactive proteins always have similar biological regulatory functions. By comparing the predicted results obtained from other methods based on blast and amino acid composition, respectively, it implies that our prediction method is quite promising that may provide an opportunity to understand this complicated pathway system well.
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A Novel Approach For Secretion Of Heterologous Proteins With Correct N-Terminal Processing By Using Α-Factor Pre Sequence In Pichia Pastoris
Lu Shen, Zhenyun Liu, Fenli Xie, Xuerui Zhang and Shuhua Tan
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00481]

Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3’end of the α-factor signal sequence pre-region, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ~ 4.7 mg L-1. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.
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Machine Learning Study Of Classifiers Trained With Biophysiochemical Properties Of Amino Acids To Predict Fibril Forming Peptide Motifs
N. V. Subba Reddy, Hareesha K. S and Smitha Sunil Kumaran Nair
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00482]

It is important to understand the cause of amyloid illnesses by predicting the short protein fragments capable of forming amyloid-like fibril motifs aiding in the discovery of sequence-targeted anti-aggregation drugs. It is extremely desirable to design computational tools to provide affordable in silico predictions owing to the limitations of molecular techniques for their identification. In this research article, we tried to study, from a machine learning perspective, the performance of several machine learning classifiers that use heterogenous features based on biochemical and biophysical properties of amino acids to discriminate between amyloidogenic and non-amyloidogenic regions in peptides. Four conventional machine learning classifiers namely  Support Vector Machine, Neural network, Decision tree and Random forest were trained and tested to find the best classifier that fits the problem domain well. Prior to classification, novel implementations of two biologically-inspired feature optimization techniques based on evolutionary algorithms and methodologies that mimic social life and a multivariate method based on projection are utilized in order to remove the unimportant and uninformative features. Among the dimenionality reduction algorithms considered under the study, prediction results show that algorithms based on evolutionary computation is the most effective. SVM best suits the problem domain in its fitment among the classifiers considered. The best classifier is also compared with an online predictor to evidence the equilibrium maintained between true positive rates and false positive rates in the proposed classifier. This exploratory study suggests that these methods are promising in providing amyloidogenity prediction and may be further extended for large-scale proteomic studies.
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Peptidoglycan Hydrolase Enterolysin A Recognizes Lipoteichoic Acid Chains In The Cell Walls Of Sensitive Bacteria
Lenka Malinicova, Katarina Dubikova, Maria Piknova, Peter Pristas and Peter Javorsky
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00483]

C-terminal domain of peptidoglycan hydrolase enterolysin A (EnlA) is involved in specific recognition and binding to the target cell envelopes and represents true cell wall binding (CWB) domain. Sensitivity/resistance to EnlA is dependent on binding ability/disability of its CWB domain. We assume that main mechanism of resistance against EnlA is absence of the specific receptor on the cell surface, which is necessary for binding of the enzyme molecule. Using competitive and enzymatic assays we have uncovered the chemical nature of the EnlA receptor, which is a lipoteichoic acid.
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Secreted Nucleobindin-2 Inhibits 3t3-L1 Adipocyte Differentiation
Yuko Tagaya, Aya Osaki, Atsuko Miura, Shuichi Okada, Kihachi Ohshima, Koshi Hashimoto, Masanobu Yamada, Tetsurou Satoh, Hiroyuki Shimizu and Masatomo Mori
[Abstract] [FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00484]

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARg, aP2, and adipsin). When Nucleobindin-2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.
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Dynamical Properties Of Steric Zipper Polymorphs Formed By A Iapp-Derived Peptide
Francesca Stanzione, Alfonso De Simone, Luciana Esposito and Luigi Vitagliano
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00485]

Understanding the molecular basis of neurodegenerative diseases have enormous implications for the development of effective therapeutic strategies. One of the most puzzling features of these pathologies is the occurrence of distinct strains, which are believed to be generated by alternative conformational transitions of the same protein/peptide. Very recently, it has been discovered that small model peptides are able to form alternative tightly packed assemblies (polymorphs) in the crystalline state. Intriguingly, it has been postulated that the different polymorphs of the same polypeptide sequence may be representative of distinct strains. As the organization of crystalline aggregates of small peptides may be heavily biased by crystal packing, we have here performed MD simulation on steric zipper polymorphs formed by of the IAPP-derived fragment SSTNVG. Our analyses show that these aggregates are rather stable also in a non-crystalline environment. This finding corroborates the hypothesis that steric zipper assemblies are good candidates to account for the phenomenon of strain in neurodegenerative diseases. Present investigations also provide clues on the factors that favour the formation of polymorphs. Indeed, the intrinsic stability of individual β-sheets formed by SSTNVG strands is very poor. Therefore, the formation of these aggregates is essentially dictated by inter-sheet interactions established within the steric zipper assembly.
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A Simple And Universal Method To Express Protein In Unfused Form
Tianwen WANG, Aitao LI, Xingyuan Mab, Guocheng DU and Jian CHEN
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00486]

The effect of additional amino acid residue(s) fused to the N- and/or C-terminal on properties of the heterogeneously expressed protein is usually difficult to be predicted. Recombinant proteins expressed without any fused sequence should be the most desired materials for related studies, such as protein drug preparation, biochemistry investigations. Here, we report a very simple and universal method enabling the expression of protein in its unfused form between the same two restriction enzyme sites (at a higher level) if a plasmid can support the fused expression. The method provided an assessable solution for unfused expression without increase in experimental resource; the necessary material is an additional primer. The method is especially useful for making whole-cell biocatalyst in which no purification steps are required.
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Ph-Sensitive Self-Associations Of The N-Terminal Domain Of Nbce1-A Suggest A Compact Conformation Under Acidic Intracellular Conditions
Harindarpal S. Gill
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00487]

NBCe1-A is an integral membrane protein that cotransports Na+ and HCO3- ions across the basolateral membrane of the proximal tubule. It is essential for maintaining a homeostatic balance of cellular and blood pH. In X-ray diffraction studies, we reported that the cytoplasmic, N-terminal domain of NBCe1-A (NtNBCe1-A) is a dimer. Here, biophysical measurements show that the dimer is in a concentration-dependent dynamic equilibrium among three additional states in solution that are characterized by its hydrodynamic properties, molar masses, emission spectra, binding properties, and stabilities as a function of pH. Under physiological conditions, dimers are in equilibrium with monomers that are pronounced at low concentration and clusters of molecular masses up to 3-5 times that of a dimer that are pronounced at high concentration. The equilibrium can be influenced so that individual dimers predominate in a taut conformation by lowering the pH. Conversely, dimers begin to relax and disassociate into an increasing population of monomers by elevating the pH. A mechanistic diagram for the inter-conversion of these states is given. The self-associations are further supported by surface plasmon resonance (SPR-Biacore) techniques that illustrate NtNBCe1-A molecules transiently bind with one another. Bicarbonate and bicarbonate-analog bisulfite appear to enhance dimerization and induce a small amount of tetramers. A model is proposed, where the Nt responds to pH or bicarbonate fluctuations inside the cell and plays a role in self-association of entire NBCe1-A molecules in the membrane.
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SOMEViz: a Web Service for Site of Metabolism Estimating and Visualizing
Qian-Cheng Shen, Ming-Yue Zheng, Jing Lu, Cheng Luo, Wei-Liang Zhu, Kai-Xian Chen, Xiao-Min Luo and Hua-Liang Jiang
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00488]

Phase I metabolism is an important consideration in drug discovery because it profoundly affects the toxicity and activity profile of a drug candidate. In these metabolic processes, CYP450 family is responsible for the majority of biotransformation events. However, it is still an important challenge to predict sites of metabolism (SOM) of a new chemical entity due to the complex reaction mechanism and variety in CYP450 enzymes. SOMEViz is an online service designed for predicting and visualizing human cytochromes P450 (CYP450)-mediated sites of metabolism (SOM) of a molecule, on the basis of a previously reported model [1]. The service provides an access for predicting sites of metabolism of molecules with reasonable accuracy, and predicted results are shown in a user-friendly as well as interactive way, which may help chemists explore metabolism properties of chemicals in the early stage of drug discovery. The web-based GUI of SOMEViz offers user a straightforward way to manage and visualize the sites of metabolism (SOM) prediction results. The service and examples are available free of charge at http://www.dddc.ac.cn/some.
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Epitope Mapping And Identification Of Amino Acids Critical For Rabbits Igg-Binding To Linear Epitopes On Buffalo Beta-Lactoglobulin
Li Xin, Chen Hongbing, Gao Jinyan, Liu Fahui and Wen Xuefang
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00489]

Buffalo milk safety was highlighted with the increase in dietary consumption, and a little information is available on buffalo milk allergy except for cross-reactivity between buffalo and cow milk. In this work, linear epitopes and critical amino acids of buffalo β-lactoglobulin were defined by 4 rabbit’s sera using SPOTTM peptide arrays approach based on the defined mimotope. The eight epitopes on buffalo β-lactoglobulin were located in the position of A6(21-30), A7(AA25-34), A8 (29-38), B4 (73-82), B5(77-86), C(87-96), F4(134-143) and F8(150-159), respectively. Among them, four epitopes (A7, A8, F4 and F8) were described as the most major epitopes and peptide (A6, B4, B5 and C) as the second major epitopes. Following single AA substitutions (Alanine or Glycine) at each position of the major epitopes, 2，3，2，3，5 and 3 of critical amino acids were identified on epitopes of A6, A8, B5, C , F4 and F8, respectively, which vary in distribution among the epitopes, such as in C terminal or N terminal and in continuous or discontinuous forms, characteristics including hydrophobicity, polar and charge, and existed frequency.
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Predicting The Classification Of Transcription Factors By Incorporating Their Binding Site Properties Into A Novel Mode Of Chou’s Pseudo Amino Acid Composition
Liang-Yun Ren, Yu-Sen Zhang and Ivan Gutman
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00490]

Transcription factors (TF) are proteins that control the first step of gene expression, the transcription of DNA into RNA sequences. The mechanism of transcriptional regulatory can be much better understood if the category of transcription factors is known. We developed a new method for predicting the classification of transcription factors by incorporating their binding site properties into a novel mode of Chou’s pseudo amino acid composition. The properties include the length of TFBSs for a TF, a new_PWM value, the proportion of not conservative TFBSs, the proportion of no-nucleosome of TFBSs, the proportion of conserved-nucleosome of TFBSs, and the GC content of TFBSs. We construct a vector with these properties to represent a TF. Then the vectors which stand for TFs were classified with SVMs. The high accuracy obtained shows that these properties are of great significance for a TF.
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Expression In Pichia Pastoris And Biological Activity Of Avian Β-Defensin 6 And Its Mutant Peptide Without Cysteines
Yanping Cao, Qingquan Ma, Anshan Shan and Na Dong
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00491]

Avian β-defensin 6 (AvBD-6) is an antimicrobial peptide that plays significant roles in the innate immunity of chickens. To explore the effects of disulfide bonds on antimicrobial activity of AvBD-6, two peptides with or without Cys residues were designed and expressed in Pichia Pastoris. The peptide AvBD-6-B was obtained by removing six Cys residues of AvBD-6. According to the codon bias of Pichia Pastoris, the genes of AvBD-6 and AvBD-6-B were synthesized. The Bgl Ⅱ-linearized recombinant plasmids pGAPHαM-AvBD-6 and pGAPHαM-AvBD-6-B were transformed into Pichia Pastoris GS115 by electroporation. The recombinant AvBD-6 and AvBD-6-B were expressed in YPD for 48 h, 72 h and 96 h at 30 °C Tricine-SDS-PAGE analysis demonstrated that both AvBD-6 and AvBD-6-B were expressed in Pichia Pastoris. The concentration of recombinant AvBD-6 and AvBD-6-B reached 114.9 mg/l and 93.8 mg/l, respectively. The expression products exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria. Antimicrobial activity of AvBD-6-B suggests that the removal of six Cys residues had no significant effect on the antimicrobial activity of avian β-defensins. Neither peptide showed hemolytic activity. This study could serve as an impetus for the production of this antimicrobial peptide as a replacement for antibiotics in animal feed.
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Purification and Characterization of a Lectin of the Swartzieae Legume Taxa
Andreia Varmes Fernandes, Márcio Viana Ramos, Ilka Maria Vasconcelos, Ana Cristina Oliveira Monteiro Moreira, Frederico Bruno Moreno, Jose Odair Pereira and José Francisco de Carvalho Gonçalves
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00492]

This work aimed at describing the first biochemical and structural data of a lectin belonging to Swartzieae, a primitive Legume Taxa. A lactose-binding seed lectin (SLL) was purified by affinity chromatography of crude saline extracts of Swartzia laevicarpa on immobilized lactose. The SLL agglutinated rabbit erythrocytes but not rat or human (A, B, O) erythrocytes. Lectin activity was retained after heating at 100 °C for 15 min and was best inhibited by N-acetylgalactosamine, lactose and galactose. The lectin exhibited a single electrophoretic pattern that corresponded to a molecular mass of 29,000 Da, which was confirmed by MS analysis. In addition, the lectin reacted positively with Schiff’s reagent. The unique N-terminal amino acid sequence (39 residues) and the internal peptide sequence were determined by Edman degradation and MS/MS, respectively. The sequencing revealed complete homology of the SLL with legume lectins belonging to primitive groups (Dalbergieae and Sophoreae). The SLL (at 1 mg/ml) did not exhibit antifungal activity against various phytopathogens or cytotoxicity (at 100 μg/ml) towards different cancer cell lines.
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A Method for Isolation of DNA-binding Proteins Based on Solubility of DNA-protein Complexes
Hua Yang, Huang Li, Li-qun Rao, Gui-you Long, Guo-ping Peng and Lu Jin
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00493]

The study of DNA-binding proteins is crucial in understanding gene regulatory networks. We developed a new method for the enrichment of DNA-binding proteins based on the variability of DNA-protein complexes’ solubility in different ionic strength solutions. 0.14M sodium chloride was determined as the most efficient extraction concentration to precipitate DNA-binding proteins. SDS-PAGE analysis revealed that some high-abundance proteins were removed effectively and at the same time DNA-binding proteins were isolated in this simple process. Twenty kinds of proteins were identified in the acquired sample by 1-D gel-LC-MS/MS. Furthermore, computerized analysis of MS data showed that quite a number of unmatched peptides have the classic structure of leucine zipper or zinc finger, which were symbolic elements of transcription factors. These results suggested that this new method can acquire DNA-binding proteins effectively and allow improvement in the isolation of high-quality DNA-binding proteins.
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L-Arginine Mediated Renaturation Enhances Yield Of Human, α6 Type Iv Collagen Non-Collagenous Domain From Bacterial Inclusion Bodies
Venugopal Gunda, Chandra Shekhar Boosani, Raj Kumar. Verma, Chittibabu Guda and Yakkanti Akul Sudhakar
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00494]

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained  using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility and purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.
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Hydropathic Self-Organized Criticality: A Magic Wand for Protein Physics
J. C. Phillips
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00495]

Self-organized criticality (SOC) is a popular concept that has been the subject of more than 3000 articles in the last 25 years.  The characteristic signature of SOC is the appearance of self-similarity (power-law scaling) in observable properties.  A characteristic observable protein property that describes protein-water interactions is the water-accessible (hydropathic) interfacial area of compacted globular protein networks.  Here we show that hydropathic power-law (size- or length-scale-dependent) exponents derived from SOC enable theory to connect standard Web-based (BLAST) short-range amino acid (aa) sequence similarities to long-range aa sequence hydrpopathic roughening form factors that hierarchically describe evolutionary trends in water - membrane protein interactions.  Our method utilizes hydropathic aa exponents that define a non-Euclidean metric realistically rooted in the atomic coordinates of 5526 protein segments. These hydropathic aa exponents thereby encapsulate universal (but previously only implicit) non-Euclidean long-range differential geometrical features of the Protein Data Bank.  These hydropathic aa exponents easily organize small mutated aa sequence differences between human and proximate species proteins.  For rhodopsin, the most studied transmembrane signaling protein associated with night vision, analysis shows that this approach separates Euclidean short- and non-Euclidean long-range aa sequence properties, and shows that they correlate with 96% success for humans, monkeys, cats, mice and rabbits.  Proper application of SOC using hydropathic aa exponents promises unprecedented simplifications of exponentially complex protein sequence-structure-function problems, both conceptual and practical.
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Proteome-Wide Inference of Human Endophilin 1-Binding Peptides
Gang Wu, Zeng-Li Zhang, Chun-Jiang Fu, Feng-Lin Lv and Fei-Fei Tian
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00496]

Human endophilin 1 (hEndo1) is a multifunctional protein that was found to bind a wide spectrum of proline-rich endocytic proteins through its Src homology 3 (SH3) domain. In order to elucidate the unknown biological functions of hEndo1, it is essential to find out the cytoplasmic components that hEndo1 recognizes and binds. However, it is too time-consuming and expensive to synthesize all peptide candidates found in the human proteome and to perform hEndo1 SH3–peptide affinity assay to identify the hEndo1-binding partners. In the present work, we describe a structure/sequence-hybrid approach to perform proteome-wide inference of human hEndo1-binding peptides using the information gained from both the primary sequence of affinity-known peptides and the interaction profile involved in hEndo1 SH3–peptide complex three-dimensional structures. Modeling results show that (i) different residue positions contribute distinctly to peptide affinity and specificity; P-1, P2 and P4 are most important, P1 and P3 are also effective, and P-3, P-2, P0, P5 and P6 are relatively insignificant, (ii) the consensus core PXXP motif is necessary but not sufficient for determining high affinity of peptides, and some other positions must be also essential in the hEndo1 SH3–peptide binding, and (iii) the alternating arrangement of polar and nonpolar amino acids along peptide sequence is critical for the high specificity of peptide recognition by hEndo1 SH3 domain. In addition, we also find that the residue type at a specific position of hEndo1-binding peptides is not stringently invariable; amino acids that possess similar polarity could replace each other without substantial influence on peptide affinity. In this way, hEndo1 presents a broad specificity in the peptide ligands that it binds.
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Characterization of the Interaction Between Endostatin Short Peptide and VEGF Receptor 3
Kyu-Yeon Han, Dimitri T. Azar,Abdellah Sabri, Hyun Lee, Sandeep Jain, Bao-Shiang Lee and Jin-Hong Chang
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00497]

Corneal angiogenesis and lymphangiogenesis areinduced by vascular endothelial growth factors (VEGFs) signaling through its receptors VEGFR-1, -2, and -3. Endostatin is a peptide antagonist of these receptors that causes inhibition of bFGF-induced corneal angiogenesis and lymphangiogenesis. Here we show that binding of VEGF-C and endostatin to recombinant VEGFR-3 is competitive. Alignments of the primary amino acid sequences of VEGF-C and the C-terminal endostatin peptide (mEP:LEQKAASCHNSYIVLCIENSFMTSFSK) identified two conserved cysteine residues separated by seven amino acids.Peptides ofVEGF-C and mEPcontaining these conserved residues bound toVEGFR-3.However, substitution of alanine for either of thecysteinesin the mEPpeptide perturbed the secondarystructure,and this mutated peptide was unable to bind to VEGFR-3. Analysis by surface plasmon resonance demonstrated that the binding of the mEP peptide forrecombinant VEGFR-3 had a Ka of 1.41×107M-1s-1, Kd of 0.6718 s-1, and a KD of 4.78×10-8M. Characterization of the mechanism of endostatin binding to VEGFR-3 may lead to the development of novel therapies for lymphangiogenesis-related disorders, such as transplant rejection, lymphedema, and cancer metastasis.
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Functional Analysis of Hybrid Peptide CAMA-Syn: Expression in Mammalian Cells and Antimicrobial Potential
Junlin Zhang, Sha Peng, Xiang Chengand Huayan Wang
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00498]

CAMA-syn, a hybrid composed of N-terminal α-helical segment of Cecropin A(amino acid 1-8) and Magainin 2 (amino acid 1-12), is a novel small peptide with the potent antibacterial and synergistic activity without cytotoxicity. In order to test the antibacterial function of CAMA-syn produced in mammalian cells, several vectors containing the synthesized CAMA-syn DNA fragment were constructed and transfected into recipient cells. The results showed that CAMA-syn fusion to green fluorescent protein (GFP) or to hemagglutinin epitope (HA) tag was expressed in both bovine embryo fibroblasts (BEF) and mouse macrophage RAW264.7 cells. The antibacterial assays of CAMA-syn were conducted against both Gram positive and negative bacteria, including S. abortusovis, P. anatis, S. hyicus and S. suis. The results of colony-forming efficiency and cell growth curves proved that the in vitro expressed CAMA-syn could have the antibacterial activity, demonstrating that macrophage specific expression of antimicrobial peptide CAMA-syn could inhibit the growth of bacteria.
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Purification Of Pectin Methylesterase From Lycopersicon Esculentum And Its Application
Shashi kant and Reena Gupta
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00499]

Pectin methylesterase (PME) (3.1.1.11) is the pectin degrading enzyme which catalyses the hydrolysis of pectin methylester group, resulting in de-esterification. PME is widely distributed in plants, fungi, yeast and bacteria. In the present study, PME was extracted from tomato by using 8.8% NaCl (4°C). The crude enzyme precipitated with 60% ammonium sulphate resulted in 1.02 fold purification of the enzyme. The purification was done by ion exchange chromatography using DEAE-Cellulose column. This resulted in 1.82 fold purification of the enzyme. The molecular weight of purified enzyme was determined by SDS-PAGE which was found to be 34.0 kDa. During characterization of the purified enzyme, the maximum activity was found at temperature 50°C, pH 6.5, reaction time 45 min. Citrus pectin was the best substrate for maximum enzyme activity. The enzyme did not require any metal ion to express its activity, enzyme was found to be very stable at 4°C and at 50°C the enzyme was stable upto 2 h as it retained 70% of its activity. The Km and Vmax values of the enzyme were found to be 0.115 mg/ml and 1.03 μmol/ml/min. PME enhance the pectin degradation process in apple juice clarification  in combination with polygalacturonase and increased %T650 from 1.7% to 5.6%.
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Crystal Structure of a Flavin-dependent Thymidylate Synthase from Helicobacter pylori strain 26695
Xiaoli Zhang, Jinyong Zhang, Gang Guo, Xuhu Mao, Yonglin Hu and Quanming Zou
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00500]

ThyX, a flavin-dependent thymidylate synthase that is involved in the synthesis of dTMP from dUMP, is a promising target for the development of novel antibacterial drugs that aimed at blocking the biosynthesis of dTMP, one of the building blocks of DNA. This enzyme has been recently identified in some dsDNA viruses and pathogenic bacteria, including the gastric pathogen Helicobacter pylori. It shares neither sequence nor structural homology with the classical ThyA in humans and other organisms. Further more, ThyX and ThyA are the only source of dTMP in these organisms and other pathways cannot substitute for their function. Thus, ThyX-specific inhibitors could be effective antibacterial reagents while having no impact on human cells. Here we report the crystal structure of ThyX from Helicobacter pylori strain 26695 in complex with co-factor FAD and substrate dUMP at 2.5 Å resolution, which consists of a 1.5 tetramer of ThyX with a total of 1248 residues, six FAD and six dUMP molecules in an asymmetric unit. The structure revealed the key residues that are involved in co-factor FAD and substrate dUMP binding, site-directed mutagenesis were performed to analysis the importance of these residues on ThyX activity by genetic complementation and FAD binding assay.
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Inactivation Kinetics of β-N-Acetyl-D-glucosaminidase from Green Crab (Scylla serrata) by Guanidinium Chloride and the Relationship between Its Enzyme Activity and Conformation
Ji-Ping Zhang, Bo Leng, Qian-Sheng Huang , Ya-Wen Yan, Xuan Liu, Qin Wang and Qing-Xi Chen
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00501]

β-N-acetyl-D-glucosaminidase (NAGase) is a major component of chitinolytic enzymes system, which plays an important role in the molting and hatching processes of marine organism. The effects of guanidinium chloride (GuHCl) on the activity of NAGase from green crab (Scylla serrata) were investigated in this study. Results show that GuHCl, at concentrations below 0.8 M, lead to reversible inactivation of the enzyme, and the IC50 was estimated to be 0.15 M. The relationship between the enzyme activity and conformation was charaterized by monitoring the change of protein fluorescence spectra. The fluorescence intensity of the enzyme decreases distinctly with increasing concentration of GuHCl, and the maximal emission peaks appear red-shifted (from 338 nm to 343 nm). The enzyme inactivation precedes conformational changes, indicating that the enzyme active site is more flexible than the whole enzyme molecule. The kinetics of inactivation has been studied using the kinetic method of the substrate reaction. The value of k+0 is larger than that of k+0, suggesting that the enzyme is protected to a certain extent by substrate during guanidine denaturation. Our results provide important new insights in marine organism culture, especially in crustacean growth.
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Design, Synthesis, and activity Evaluation of a new 5-Fluorouracil Prodrug Containing an Asn-Gly-Arg(NO2)COOCH3 Tripeptide
Yepeng Luan, Fanbo Jing, Jian Zhang, Mingming Zou, Xuejian Wang, Yuping Jia, , Ning Liu, Jiajia Mou and Wenfang Xu
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00502]

Aminopeptidase N (APN/CD13) of the M1 family is a broad specificity enzyme that is intimately involved in tumor angiogenesis and metastasis. Asparagine–glycine–arginine (NGR) is a tumor-homing tripeptide, which can selectively combine with APN/CD13 that is overexpressed on the surface of some tumor cells. Various anti-tumor drugs can be conjugated to NGR to improve selectivity, efficacy, and to decrease drug toxicity. In this study, a tripeptide NGR(NO2) was synthesized and conjugated with 5-fluorouracil. The anti-tumor activities of this new prodrug were evaluated in vivo. More significant anti-tumor effects were observed over the parent 5-fluorouracil.
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Bacterial Lipopolysaccharides in Plant and Mammalian Innate Immunity
Cristina De Castro, Otto Holst, Rosa Lanzetta, Michelangelo Parrilli  and Antonio Molinaro
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00504]

This mini-review gives a structural view on the lipopolysaccharides (LPSs), the endotoxin from Gram negative bacteria, paying attention on the features that are relevant for their activity as elicitors of the innate immune system of humans, animals and plants.
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Impact of Structural Domains of the Heparin Binding Hemagglutinin of Mycobacterium tuberculosis on Function
Giovanni Delogu, Giovanni Fadda and Michael J. Brennan
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00505]

Among the few well characterized virulence factors of Mycobacterium tuberculosis (Mtb) is the heparin-binding hemagglutinin (HBHA).  HBHA is a 21-kDa protein that localizes to the mycobacterial surface where it can interact with host components.  Interaction with epithelial cells and components of the extracellular matrix is mediated by the methylated lysine-rich C-terminal domain of the protein.  The N-terminal end of HBHA contains a coiled coil motif which is involved in protein oligomerization and bacterial-bacterial aggregation.  In this report, we will focus our attention on what is known about the structure of the HBHA protein and the protein function and role in TB pathogenesis.
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Resuscitation-Promoting Factors (RPF): In Search of Inhibitors
Arseny S. Kaprelyants, Galina V. Mukamolova, Alessia Ruggiero, Vadim A. Makarov, Galina R. Demina, Margarita O. Shleeva, Vasilii D. Potapov and Pavel A. Shramko
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00506]

Resuscitation promoting factors (Rpf) are a family of proteins secreted by actively growing actinobacteria, including Mycobacterium tuberculosis. Experimental evidence suggests that Rpfs play a distinct role in bacterial resuscitation and re-growth as well as reactivation of chronic  2.
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Structural And Dynamic Properties Of Incomplete Immunoglobulin-Like Fold Domains
Rita Berisio, Luciano Ciccarelli , Flavia Squeglia, Alfonso De Simone and Luigi Vitagliano
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00507]

The immunoglobulin fold (Ig-fold) is a widespread structural motif that is detected in a variety of proteins involved in diversified biological processes. The Ig-fold contains 70-110 residues that are assembled in a characteristic sandwich-like structure formed by two facing β-sheets each made of antiparallel β-strands. A number of variations on this common theme have been detected and described (Ig-like fold).  One of the most intriguing variants is characterized by the lack of a strand compared to the canonical motif (incomplete Ig-like fold). Interestingly, proteins exhibiting incomplete Ig-like fold have been shown to play an important role in mediating either protein-protein or domain-domain interactions. Protein-protein interactions mediated by incomplete Ig-like folds play a key structural role in the chaperone–usher pathway, a process that generates multi-protein assemblies essential for the adhesion of gram negative bacteria. Domains with incomplete Ig-like fold have also been discovered in the mechanism of action of adhesins belonging to the family of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Recently, a stable incomplete Ig-like fold has been detected in the peptidoglycan-binding extra-cellular portion of Staphylococcus aureus PrkC, an important Ser/Thr membrane kinase involved in bacterial growth and revival from latency. It is important to note that the occurrence of proteins with incomplete Ig-like fold is often related to cell adhesion and infectivity of bacterial pathological agents. We here report a survey of the structural data available on this peculiar structural motif highlighting analogies and differences of incomplete Ig-like fold involved in  different processes. The dynamical behavior of these domains, investigated by molecular dynamics techniques, will be also commented.
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Eight Stranded β-barrel and Related Outer Membrane Proteins: Role in Bacterial Pathogenesis
Siobhán McClean
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00508]

Gram negative bacteria have evolved many mechanisms of attaching to and invading host epithelial and immune cells.  In particular, many outer membrane proteins (OMPs) are involved in this initial interaction between the pathogen and their host.  This review focuses on a number of small pore-forming OMPs that are all composed of eight-stranded β-barrel proteins and include members of the OmpA, OmpW and OmpX families of proteins.   These proteins, together with the related OmpA-like peptidoglycan associated lipoproteins, are involved in interactions with host cells and are mediators of virulence.   In many cases, these proteins interact with host immune cells and can be considered as pathogen associated molecular patterns (PAMPS) due to their ability to signal via Toll like receptor molecules and other pattern recognition receptors.   The role of these proteins in pathogenesis is discussed here, together with the potential for these proteins to be used as immunoprophylactic agents to protect against infection.
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Isolation and Identification of Novel Neutrophil-Activating Cryptides Hidden in Mitochondrial Cytochrome c
Yoshinori Hokari, Tetsuo Seki, Hiroko Nakano, Yuko Matsuo, Akiyoshi Fukamizu, Eisuke Munekata, Yoshiaki Kiso and Hidehito Mukai
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00522]

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification. Here, we isolated and purified a novel neutrophil-activating peptide from porcine hearts, which we showed by structural analyses to have an identical primary structure to porcine mitochondrial cytochromes c (68-85). We named this novel functional octadecapeptide as mitocryptide-CYC (MCT-CYC). Structure-activity relationships of cytochrome c on β-hexosaminidase (β-HA) release from neutrophilic-differentiated HL-60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced β-HA release and that cytochromes c (70-85) was the most potent cryptide among them. Since cytochrome c is known to be involved in the apoptotic process, our results suggest that cryptides, including MCT-CYC, derived from mitochondrial cytochrome c are possible factors that induce scavenging of toxic debris produced from apoptotic cells by neutrophils.
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Local Flexibility Facilitates Oxidization of Buried Methionine Residues
Kuiran Xu, Vladimir N. Uversky and Bin Xue
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00523]

In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.
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Detection of Antibodies against Synthetic Peptides Mimicking Ureases Fragments in Sera of Rheumatoid Arthritis Patients
Iwona Konieczna, Marek Kwinkowski, Beata Kolesinska, Zbigniew Kaminski, Justyna Fraczyk, Paulina Zarnowiec and Wieslaw Kaca
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00524]

Rheumatoid arthritis (RA) is a chronic disease with an autoimmunological background. RA is mostly characterized by systemic inflammation and injuries of synovial joints. There is a hypothesis that bacterial infections may be connected with development of the disease. It has been suggested that molecular mimicry between bacterial and human antigens may be one of possible mechanisms of RA development. One of potential antigens involved in this mechanism is urease – enzyme with high structural conservatism, occurring in pathogenic and commensal bacteria. We found that the level of antibodies against peptide mimicking urease “flap” region is significantly higher in sera from patients with rheumatoid arthritis in comparison with volunteer blood donor sera. We also observed that antibodies present in RA sera may bind not only specific peptide antigens but also peptides with a slightly different structure.
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Biochemical property and in vivo efficacies of novel Val/Arg-rich antimicrobial peptide
Qing-Quan Ma, Na Dong, An-Shan Shan , Liang Wang, Wan-Ning Hu and Wen-Yu Sun
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00525]

A novel α -helical antimicrobial peptide G6 rich in Val/Arg residues has been screened previously. In this study, we further evaluated the biochemical stability, interaction with whole bacteria, and in vivo therapeutic or prophylactic role of the peptide in Salmonella typhimurium-infected mice. The results showed that G6 exhibited strong resistance to pH, heat, and salts. But G6 lost the antimicrobial activity when treated with proteolytic enzymes. G6 had no toxicity on mammalian cell. An intraperitoneal model of sepsis caused by Salmonella typhimurium was established in mice. G6 was administered intraperitoneally 1 h before or after mice were infected with Salmonella typhimurium. For the mice given peptide post-bacterial infection, the mortality of the mice and the peritoneal bacterial counts were significantly lower in the groups that were administered 2.5 mg/kg BW and 5.0 mg/kg BW of G6 (P < 0.05) compared to the PBS-treated group. Similar trend was obtained when G6 was given 1 h prior to Salmonella typhimurium infection. Peptide-membrane experiments showed that G6 was effective in permeabilizing the outer and inner membrane in a dose dependent manner, indicating that the peptide targets the cell membrane. Taken together, the results revealed that the peptide G6 may provide a useful alternative to antibiotic agents to treat or prevent bacterial infections.
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Novel design of short antimicrobial peptides derived from the bactericidal domain of avian ß-defensin-4
Na Dong, Qing-Quan Ma, An-Shan Shan, Yin-Feng Lv, Wanning Hu, Yao Gu and Yu-Zhi Li
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00526]

Short antimicrobial peptides were designed and synthesized by C-terminal truncation and residue substitution of avian ß-defensin-4. The biological activity of these peptides was examined to elucidate the structure-function relationships and find a lead peptide for the development of a novel antimicrobial peptide. The results showed that the truncation of the avian ß-defensin-4 eliminated the hemolysis of the peptide. The GLI13 derivative, developed by substituting the Cys of the truncated peptide with Ile, led to increased antimicrobial activity. These results suggest that the peptides with antimicrobial activity can be derived by truncating the avian ß-defensin-4. We further developed the GLI13 derivative with an increased net charge by residue substitution. An increase in the net charge from 4 to 8 did not result in the improvement of antimicrobial potency. Membrane-simulating experiments showed that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. A membrane depolarization assay showed that GLI13-5 killed bacteria by targeting the cytoplasmic membrane. These results suggest that the short peptide developed by truncation of linear ß-defensin may be a promising candidate for future antibacterial agents.
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Crystal structure of the Tum1 protein from the yeast Saccharomyces cerevisiae
Rui Qiu, Fengbin Wang, Meiruo Liu, Tiantian Lou and Chaoneng Ji
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00527]

Yeast tRNA-thiouridine modification protein 1 (Tum1) plays essential role in the sulfur transfer process of Urm1 system, which in turn is involved in many important cellular processes. In the rhodanese-like domain (RLD), conserved cysteine residue is proved to be the centre of active site of sulfurtransferases and crucial for the substrate recognition. In this report, we describe the crystal structure of Tum1 protein at 1.90 Å resolution which, despite consisting of two RLDs, has only one conserved cysteine residue in the C-terminal RLD. An unaccounted electron density is found near the active site, which might point to the new cofactor in the sulfur transfer mechanism.
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Biochemical characterization of thymidine monophosphate kinase from white spot syndrome virus: A functional domain from the viral ORF454
Eduardo Guevara-Hernandez, Karina D. Garcia-Orozco and Rogerio R. Sotelo-Mundo
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00528]

Nucleotide phosphorylation is a key step towards DNA replication and during viral infections the maintenance of the nucleotide triphosphates pool is required. Deoxythymidine triphosphate (dTTP) is the unique nucleotide that is produced either by de novo or salvage pathways. Thymidine monophosphate kinase (TMK) is the enzyme that phosphorylatesdeoxythymidine monophosphate (dTMP) using adenosine triphosphate (ATP) as a phosphate group donor in presence of Mg2+ yielding deoxythymidinediphosphate (dTDP) and adenosine diphosphate. The TMK region of the WSSV TK-TMK chimeric protein was overexpressed and purified. This recombinant proteinhad TMK activity, this is that dTMP was phosphorylated to dTDP and we found that the dimeric state of the protein was the functional and a theoretical structural model was built as such. Future work will focus towards a structural characterization as an antiviral target.
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Aqueous Microwave-Assisted Solid-Phase Peptide Synthesis Using Fmoc Strategy: In-Water Synthesis of “Difficult Sequences”
Keiko Hojo, Hideki Ichikawa, Asaki Hara, Mare Onishi, Koichi Kawasaki and Yoshinobu Fukumori
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00529]

A microwave assisted peptide synthesis in water using nanosized Fmoc-amino acids was developed. 5, 7, and 10 mer peptides (Leu-enkephalinamide, dermorphinamide, and a typical difficult sequence, ACP (65-74) peptide) were successfully synthesized in water according to Fmoc chemistry using water-dispersible nanoparticles with microwave irradiation.
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Characterization of Glycine Substitution Mutations within the Putative NAD+-binding Site of Bacillus licheniformis Aldehyde Dehydrogenase
Yen-Chung Lee, Den-Tai Lin, Hsiang-Ling Chen, Huei-Fen Lo, Hui-Yu Hu, Nai-Wan Hsiao and Long-Liu Lin
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00530]

The NAD+-requiring enzymes of the aldehyde dehydrogenase (ALDH) family contain a glycine motif, GX1–2GXXG, which is reminiscent of the fingerprint region of the Rossman fold, a conserved structural motif of the classical nicotinamide nucleotide-binding proteins. In this research, the role of three glycine residues situated within the putative NAD+-binding motif (211-GPGSSAG) together with Gly233 and Gly238 of Bacillus licheniformis ALDH (BlALDH) were probed by site-directed mutatgenesis. Fifteen mutant BlALDHs were obtained by substitution of the indicated glycine residues with alanine, glutamate and arginine. Except for the Ala replacement at positions 211, 213, 217 and 238, the remaining mutant enzymes lost the dehydrogenase activity completely. Tryptophan fluorescence and far-UV circular dichroism spectra allowed us to discriminate BlALDH and the inactive mutant enzymes, and unfolding analyses further revealed that they had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlALDH and the functional variants had a comparable Tm value, but the value was reduced by more than 5.1°C in the rest of mutant enzymes. Acrylamide quenching analysis showed that the inactive mutant enzymes had a dynamic quenching constant greater than that of BlALDH. Native BlALDH started to unfold beyond ~ 0.21 M GdnHCl and reached an unfolded intermediate, [GdnHCl]0.5, N-U, at 0.92 M equivalent to free energy change ( ) of 12.34 kcal/mol for the N → U process, whereas the denaturation midpoints for mutant enzymes were 0.45–1.61 M equivalent to of 0.31–4.35 kcal/mol. Taken together, these results strongly suggest that the explored glycines are indeed important for the catalytic activity and structural stability of BlALDH.
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Local Flexibility Facilitates Oxidization of Buried Methionine Residues
Kuiran Xu, Vladimir N. Uversky and Bin Xue
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00531]

In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.
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Principles, Challenges and Advances in ab initio Protein Structure Prediction
Arunachalam Jothi
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00532]

The gap between known protein sequences and structures is increasing rapidly and experimental methods alone will not be able to fill in this gap. Therefore it is necessary to use computational methods to predict protein structures. Template based modeling methods could be used for sequences, which have detectable relationship with sequences of one or more experimentally determined protein structures. For predicting the structure of proteins, which does not share a detectable sequence relationship with experimental structures, ab initio protein structure prediction techniques must be used. The methods under ab initio protein structure prediction category aim to predict the structure of a protein from the sequence information alone, without any explicit use of previously known structures. These methods use thermodynamic principles and try to identify the native structure of a protein as the global minimum of a potential energy landscape. However, such methods are computationally complex and are extraordinarily challenging. There has been significant progress in the development of ab inito protein structure prediction methods over the past few years. This review describes the basic principles, the complexity, challenges and recent progresses of ab initio protein structure prediction.
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SVM prediction of ligand-binding sites in bacterial lipoproteins employing shape and physio-chemical descriptors
Kiran Kadam, Prashant Prabhakar and V. K. Jayaraman
[FULL-TEXT INQUIRY] [BSP/PPL/E-Pub/00533]

Bacterial lipoproteins play critical roles in various physiological processes including the maintenance of pathogenicity and numbers of them are being considered as potential candidates for generating novel vaccines. In this work, we put forth an algorithm to identify and predict ligand-binding sites in bacterial lipoproteins. The method uses three types of pocket descrip¬tors, namely fpocket descriptors, 3D Zernike descriptors and shell descriptors, and combines them with Support Vector Machine (SVM) method for the classification. The three types of descriptors represent shape-based properties of the pocket as well as its local physio-chemical features. All three types of descriptors, along with their hybrid combinations are evaluated with SVM and to improve classification performance, WEKA-InfoGain feature selection is ap¬plied. Results obtained in the study show that the classifier successfully differentiates between ligand-binding and non-binding pockets. For the combination of three types of descriptors, 10 fold cross-validation accuracy of 86.83% is obtained for training while the selected model achieved test Matthews Correlation Coefficient (MCC) of 0.534. Individually or in combina¬tion with new and existing methods, our model can be a very useful tool for the prediction of potential ligand-binding sites in bacterial lipoproteins.
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