Protein & Peptide Letters

ISSN: 0929-8665

Protein & Peptide Letters
Volume 13, Number 8, 2006

Contents


Regular Papers

Purification and Structural Characterization of Human ERp29 Pp. 753-759
J. Zheng, X. Liu, X. Yan, L. Dai and C. Ji
[Abstract] [Purchase Article]


A Simple Search of TM Segments in Polytopic Membrane Protein Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Pp. 761-767
Y. Abe, T. Hamasaki, S. Turusaki, S. Takazaki, J. Xiui, D. Kang and N. Hamasaki
[Abstract] [Purchase Article]


The Stability Curve of Hen Egg White Lysozyme Pp. 769-772
S.S. Younvanich and B.M. Britt
[Abstract] [Purchase Article]


Quantitative Analysis of Cytochrome C Released from Rice Mitochondria Using the Adsorptive Polarographic Wave of Guanidine Modified Co(II)-Cytochrome C Complex Pp. 773-777
C. Xia, X. Liu, D. Luo, H. Wang, F. He, L. Yuan and C. Wang
[Abstract] [Purchase Article]


Isolation and Partial Characterization of Ribonuclease Inhibitor from Goat Liver Pp. 779-783
J. Chatterjee, T.K. Maiti and S. Dasgupta
[Abstract] [Purchase Article]


High-Level Expression and Purification of Human Epidermal Growth Factor with SUMO Fusion in Escherichia coli Pp. 785-792
Z. Su, Y. Huang, Q. Zhou, Z. Wu, X. Wu, Q. Zheng, C. Ding and X. Li
[Abstract] [Purchase Article]


Effects of Physical, Ionic, and Structural Factors on the Binding of Repressor of Mycobacteriophage L1 to Its Cognate Operator DNA Pp. 793-798
T. Ganguly, P.K. Chanda, A. Bandhu, P. Chattoraj, M. Das and S. Sau
[Abstract] [Purchase Article]


Purification and Thermal Characterization of a Novel Peroxidase from a Local Chick Pea Cultivar Pp. 799-804
H.N. Bhatti, A. Najma, M. Asgher, M.A. Hanif and M.A. Zia
[Abstract] [Purchase Article]


Antimicrobial Properties of Nuclear Diffusion Inhibitory Signal of HIV-1 Rev Pp. 805-806
N. Kobayashi and T. Yoshida
[Abstract] [Purchase Article]


In Silico Criterion for Prediction of Effects of p53 Gene Missense Mutations on p53-Mdm2 Feedback Loop Pp. 807-814
N. Veljkovic and V. Perovic
[Abstract] [Purchase Article]


N-Terminal Mutational Analysis of the Interaction Between Growth-Blocking Peptide (GBP) and Receptor of Insect Immune Cells Pp. 815-822
S. Watanabe, M. Tada, T. Aizawa, M. Yoshida, T. Sugaya, M. Taguchi, T. Kouno, T. Nakamura, M. Mizuguchi, M. Demura, Y. Hayakawa and K. Kawano
[Abstract] [Purchase Article]


Expression and Purification of Exendin-4 Dimer in Escherichia coli and Its Interaction with GLP-1 Receptor In Vitro Pp. 823-827
L. Yi, X. Yin, D. Wei and Y. Ma
[Abstract] [Purchase Article]


Transformation of a Biologically Active Peptide into Peptoid Analogs While Retaining Biological Activity Pp. 829-833
B. Hoffmann, T. Ast, T. Polakowski, U. Reineke and R. Volkmer
[Abstract] [Purchase Article]


Allergenic Tropomyosins and Their Cross-Reactivities Pp. 835-845
K.Y. Jeong, C.-S. Hong and T.-S. Yong
[Abstract] [Purchase Article]


Polyproline Helices in Protein Structures: A Statistical Survey Pp. 847-854
R. Berisio, S. Loguercio, A. De Simone, A. Zagari and L. Vitagliano
[Abstract] [Purchase Article]


Crystallization Reports


Crystallization and Preliminary Crystallographic Analysis of Allograft Inflammatory Factor 1 Pp. 855-858
S. Li, X. Liao, Z. Chen and M. Bartlam
[Abstract] [Purchase Article]


Expression, Purification, Crystallization, and Preliminary X-Ray Analysis of the Human UDP-Glucose Dehydrogenase Pp. 859-862
J.W. Huh, R.C. Robinson, H.S. Lee, J.I. Lee, Y.S. Heo, H.T. Kim, H.J. Lee, S.W. Cho and H. Choe
[Abstract] [Purchase Article]




Abstracts

[Back to top] [Purchase Article]
Purification and Structural Characterization of Human ERp29
J. Zheng, X. Liu, X. Yan, L. Dai and C. Ji

ERp29 is a major resident of the endoplasmic reticulum (ER) and is postulated to play an important molecular chaperone role in most animal cells. Human ERp29 was isolated to homogeneity in high yield by using a bacterial expression system. Its secondary structure was studied by circular dichroism (CD), Fourier transformed infrared spectroscopy (FTIR) and Raman spectroscopy and it was found that human ERp29 comprises significant _-helical structure. The details of its temperature-induced conformational changes was studied by CD and FTIR for the first time, revealing that the protein is stable below 50 °C and has two distinct structural transitions between 50 °C and 70 °C. This may shed light on ERp29’s inability to protect substrate proteins against thermal aggregation.


[Back to top] [Purchase Article]
A Simple Search of TM Segments in Polytopic Membrane Protein Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
Y. Abe, T. Hamasaki, S. Turusaki, S. Takazaki, J. Xiui, D. Kang and N. Hamasaki

Using both high performance liquid chromatography (HPLC) and amino acid sequencing (AAS), we previously analyzed band 3 TM peptide-segments that make up the transmembrane protein structure. However, the HPLC/AAS combination method was highly time-consuming. Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry is used to obtain accurate molecular weight information for proteins/peptides simply and sensitively. We applied the MALDI-TOF mass spectrometry technique to search for TM segments in membrane proteins. In combination with trypsin cleavages after alkali treatments (pH12 or 13) and sample preparation using organic solvents for MALDI-TOF mass spectrometry, we determined the TM segments of band 3 and glycophorin A in erythrocyte membrane. The method can be applied to other polytopic membrane proteins in erythrocyte membrane.


[Back to top] [Purchase Article]
The Stability Curve of Hen Egg White Lysozyme
S.S. Younvanich and B.M. Britt

The physiological stability curve -- a plot of the free energy of unfolding versus temperature -- is calculated for hen egg white lysozyme from a combination of extrapolated unfolding thermodynamic data from reversible conditions and isothermal titrations with guanidine hydrochloride. The shape of the curve suggests the existence of only one folded conformation.


[Back to top] [Purchase Article]
Quantitative Analysis of Cytochrome C Released from Rice Mitochondria Using the Adsorptive Polarographic Wave of Guanidine Modified Co(II)-Cytochrome C Complex
C. Xia, X. Liu, D. Luo, H. Wang, F. He, L. Yuan and C. Wang

A new, simple and sensitive method for the quantitative analysis of cytochrome C (Cyt C) based on the reduction wave of guanidine modified Co(II)-Cyt C complex at about –1.74 V (vs. SCE) by single sweep polarography in the solution containing 8×10^-6 mol L^-1 CoCl₂, 0.04 mol L^-1 guanidine hydrochloride, 0.2 mol L^-1 NaOH and 0.5% Na₂SO₃. The peak height is linearly proportional to the concentration of Cyt C in the range of 0.005~1.500 mg L^-1 (correlation coefficient 0.999). Common amino acids, saccharide, organic acid and metal ions of appropriate concentrations have no interference on the Cyt C determination. The released Cyt C in the process of mitochondrial permeability transition of Hong-Lian cytoplasmic male sterile line of rice has been measured by the method, and the result is satisfactory


[Back to top] [Purchase Article]
Isolation and Partial Characterization of Ribonuclease Inhibitor from Goat Liver
J. Chatterjee, T.K. Maiti and S. Dasgupta

Ribonuclease inhibitor (RI), a 50 kDa protein, has been found both in mammalian and nonmammalian tissues. We have isolated RI from goat liver and partial characterization has been accomplished. For the isolation of RI, DEAE cellulose column chromatography followed by affinity chromatography using CNBr activated Sepharose 4B was performed. The inhibition of ribonucleolytic activity of Ribonuclease A has been checked by an agarose gel based assay. The antiangiogenic property of the protein was tested by the chorioallantoic membrane (CAM) assay. Results indicate inhibition of angiogenesis.


[Back to top] [Purchase Article]
High-Level Expression and Purification of Human Epidermal Growth Factor with SUMO Fusion in Escherichia coli
Z. Su, Y. Huang, Q. Zhou, Z. Wu, X. Wu, Q. Zheng, C. Ding and X. Li

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.


[Back to top] [Purchase Article]
Effects of Physical, Ionic, and Structural Factors on the Binding of Repressor of Mycobacteriophage L1 to Its Cognate Operator DNA
T. Ganguly, P.K. Chanda, A. Bandhu, P. Chattoraj, M. Das and S. Sau

To determine the factors influencing the binding of L1 repressor to its cognate operator DNA, several gel shift as well as bioinformatic analyses have been carried out. The data show that time, temperature, salt, and pH each greatly affect the binding. In order to achieve optimum operator binding of L1 repressor in Tris buffer, the minimum requirements of time, temperature, salt, and pH were estimated to be 1 min, 32°C, NaCl (50 mM), and 7.9, respectively. Interestingly Na⁺ but not NH₄⁺, K⁺, or Li⁺ was found to augment significantly the binding activity of CI protein above the basal level. Anions like Cl^-, citrate^-, acetate^-, and H₂PO₄^- do not alter the binding of L1 repressor to its operator. We also show that an in frame deletion mutant of L1 repressor which does not carry the putative HTH motif (at its N-terminal end) fails to bind to its cognate operator DNA even at very high concentrations. The putative HTH motif was found highly conserved and evolutionarily very close to that of regulatory proteins of Y. pestis, H. marismortui, A. tumefaciens, etc. Taken together we suggest that N-terminal end of L1 repressor carries a HTH motif. Further analysis of the putative secondary structures of mycobacteriophage repressors reveals that two common regions encompassing more than 90% of primary sequence are present in all the four repressor molecules studied here. The results suggest that these common regions are utilized for carrying out identical functions.


[Back to top] [Purchase Article]
Purification and Thermal Characterization of a Novel Peroxidase from a Local Chick Pea Cultivar
H.N. Bhatti, A. Najma, M. Asgher, M.A. Hanif and M.A. Zia

A novel peroxidase isolated from a local chick pea (Cicer arietinum L.) cultivar (Balksar 2000) was purified by means of ammonium sulfate precipitation, DEAE-cellulose chromatography and two runs on gel filtration. The purified enzyme has a specific activity of 2045 U/mg with 17 % activity recovery. The molecular mass of the enzyme was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. Optimum pH and temperature of the enzyme were 5.5 and 45°C respectively. The thermal denaturation of local chick pea peroxidase was studied in aqueous solution at temperatures ranging from 45°C to 65°C. The temperature of 50% inactivation of the enzyme was found to be 68°C. The enthalpy (ΔH*) and free energy (ΔG*) of thermal denaturation of chick pea peroxidase were 101.4 and 103.4 k J/mol respectively at 65°C.Metals like Zn²⁺, Mn²⁺, Hg²⁺, Co²⁺ and Al³⁺ slightly inhibited the peroxidase activity while Ca²⁺, Mg²⁺ and Ba²⁺ have no effect on enzyme activity. The high specific activity and thermal stability make chick pea peroxidase an alternative to horseradish peroxidase (HRP) in various applications.

[Back to top] [Purchase Article]
Antimicrobial Properties of Nuclear Diffusion Inhibitory Signal of HIV-1 Rev
N. Kobayashi and T. Yoshida

Nuclear Diffusion Inhibitory Signal (NIS) has been identified in human immunodeficiency virus type 1 (HIV-1) Rev as a nuclear signal peptide which modulates nucleocytoplasmic protein trafficking and intracellular stability of the HIV-1 Rev. In this study, it was discovered that antimicrobial properties are inherent in the NIS. This is a significant finding that the NIS, which does not exist solely for self defense, in fact possesses antimicrobial properties.


[Back to top] [Purchase Article]
In Silico Criterion for Prediction of Effects of p53 Gene Missense Mutations on p53-Mdm2 Feedback Loop
N. Veljkovic and V. Perovic

The Informational Spectrum Method (ISM) is the tool for the in silico analysis of proteins which interprets protein sequence linear information using signal analyses methods. In this paper the ISM was employed to characterize the products of genetic variants of tumor suppressor gene p53 and its natural binding regulator protein Mdm2. Based on this we propose the criterion for identification of missense mutations that have impact on the p53-Mdm2 feedback loop. The efficiency of the proposed criterion was confirmed by the ISM analyses of p53 mutants reported in: (i) healthy individuals, (ii) germline mutations database and (iii) somatic mutations database.


[Back to top] [Purchase Article]
N-Terminal Mutational Analysis of the Interaction Between Growth-Blocking Peptide (GBP) and Receptor of Insect Immune Cells
S. Watanabe, M. Tada, T. Aizawa, M. Yoshida, T. Sugaya, M. Taguchi, T. Kouno, T. Nakamura, M. Mizuguchi, M. Demura, Y. Hayakawa and K. Kawano

GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The ¹H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core β–sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn². Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.


[Back to top] [Purchase Article]
Expression and Purification of Exendin-4 Dimer in Escherichia coli and Its Interaction with GLP-1 Receptor In Vitro
L. Yi, X. Yin, D. Wei and Y. Ma

Exendin-4 is a 39 amino acid peptide isolated from the Gila monster salivary gland. It is 53% homologous to GLP-1 and exhibits similar glucoregulatory activities. In this study, exendin-4 dimer (D-Ex4) was constructed, cloned into plasmid pET32a(+) and expressed in E. coli BL21(DE3). The fusion protein with His-tag at the N-terminus was purified with a Ni-NTA-agarose column. After proteolytic cleavage, D-Ex4 peptide with high purity was obtained by HPLC. The results obtained by chemical cross-linking showed that D-Ex4 maintained affinity to GLP-1 receptor.


[Back to top] [Purchase Article]
Transformation of a Biologically Active Peptide into Peptoid Analogs While Retaining Biological Activity
B. Hoffmann, T. Ast, T. Polakowski, U. Reineke and R. Volkmer

We report the stepwise transformation of a linear peptide epitope recognized by the anti-transforming growth factor α monoclonal antibody Tab2 into peptomers and finally into peptoid analogs. The key experiment in this study is the substitution analysis in which each position of the peptide is exchanged by a set of different peptoid building blocks resulting in a peptidomimetic array. After probing the array toward antibody binding, the best binding peptomer spots were selected and subjected to a successive transformation. The best peptoid found in this study has a K_D of 200 nM when binding to Tab2, which is only 8-fold higher than the starting peptide. Moreover, this approach permits to ask directly questions about the transformation of peptide lead structures into non-peptidic compounds in the context of protein recognition.


[Back to top] [Purchase Article]
Allergenic Tropomyosins and Their Cross-Reactivities
K.Y. Jeong, C.-S. Hong and T.-S. Yong

The ingestion or inhalation of some proteins may lead to adverse immune reactions. Allergens may trigger allergic reactions in genetically predisposed individuals when they are absorbed through the skin or make contact with mucous membranes. An allergic disease often deteriorates the quality of life and may sometimes be life-threatening due to anaphylactic shock.

A number of allergens have been characterized from various multicellular organisms to date. It is thought to be reasonable to pay a special attention to the substance which is highly cross-reactive and which causes adverse responses in the molecules that are not sensitized but similar to the sensitized allergen.

Tropomyosin has been described as an important food allergen in shrimp, lobster, crab, oysters, squid, and other invertebrates. Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins among invertebrates, but vertebrate tropomyosins are not known to be allergenic. Several tropomyosins from domestic arthropods have been reported to be allergenic. Recently, it was suggested that an infection of helminthic parasites might lead to sensitization to tropomyosin and elicit allergic reactions to other invertebrates.

Much effort has been made to characterize these allergenic tropomyosins from various sources. We will discuss the physicochemical characteristics and the potential application of tropomyosin for the diagnosis and therapeutics of allergic disorders.


[Back to top] [Purchase Article]
Polyproline Helices in Protein Structures: A Statistical Survey
R. Berisio, S. Loguercio, A. De Simone, A. Zagari and L. Vitagliano

A statistical survey of polyproline II (PPII) helices extracted from protein crystal structures is here reported. The average hydrophobicity of these helices is intermediate between those displayed by β-strands and coil regions and is similar to that of α-helices. PPII helices with amphipathic properties have been identified and classified. Amino acid propensities for PPII helices derived in this study differ significantly from those previously reported. They show a little albeit significant correlation with propensities for α-helices whereas they are fully non-correlated to propensities for β-sheets. Finally, PPII propensities have been correlated with amino acid frequencies in structural proteins, such as collagen and extensins.


[Back to top] [Purchase Article]
Crystallization and Preliminary Crystallographic Analysis of Allograft Inflammatory Factor 1
S. Li, X. Liao, Z. Chen and M. Bartlam

Allograft inflammatory factor-1 (AIF-1) is a 17-kDa IFN-γ inducible Ca²⁺-binding EF-hand protein involved in immune dysfunction and smooth muscle cell activation. AIF-1 was solubly expressed in E. coli and purified. Crystals of AIF-1 were grown at 291 K using PEG-8000 as precipitant. Diffraction by the AIF-1 crystal extends to 3.3 Å resolution, and the crystal belongs to the space group P4₃ with unit cell parameters a=b=73.4, c=49.1 Å.


[Back to top] [Purchase Article]
Expression, Purification, Crystallization, and Preliminary X-Ray Analysis of the Human UDP-Glucose Dehydrogenase
J.W. Huh, R.C. Robinson, H.S. Lee, J.I. Lee, Y.S. Heo, H.T. Kim, H.J. Lee, S.W. Cho and H. Choe

UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis of UDP-glucuronic acid from UDP-glucose resulting in the formation of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers. Here, human UGDH (hUGDH) was purified and crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data were collected to a resolution of 2.8 Å. The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 Å, and α = β = γ = 90.0°. Based on preliminary analysis of the diffraction data, we propose that the biological unit of hUGDH is a tetramer.