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Protein
& Peptide Letters
ISSN: 0929-8665
Protein &
Peptide Letters
Volume 18, Number 6, 2011
Contents
Study of Prolactin Permeation Through
the Pericardium and Its Bioavailability Pp. 540-543
Barbara Dolinska, Artur Caban, Lucyna Leszczynska, Grzegorz
Oczkowicz and Florian Ryszka
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The Effect of Deleting a Putative Salt Bridge on the
Properties of the Thermostable Subtilisin-Like Proteinase,
Aqualysin I Pp. 545-551
Jóhanna Arnórsdóttir, Manuela Magnúsdóttir, Ólafur H.
Friðjónsson and Magnús M. Kristjánsson
[Abstract] [Purchase
Article]
Using Pseudo Amino Acid Composition to Predict
Protease Families by Incorporating a Series of Protein Biological
Features Pp. 552-558
Lele Hu, Lulu Zheng, Zhiwen Wang, Bing Li and
Lei Liu
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Article]
Computer-Based Comparison of Structural Features
of Envelope Protein of Alkhurma Hemorrhagic Fever Virus with
the Homologous Proteins of Two Closest Viruses Pp.
559-567
Hassan Mohabatkar
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Article]
A Novel Rhodobacter sphaeroides Expression
System for Real-Time Evaluation of Heterologous Protein Expression
Levels Pp. 568-572
Zhiping Zhao, Zongli Hu, Xin Nie, Lijing Cheng, Guolin
Ding, Min Luo, Yu Pan, Yan Liang and Guoping Chen
[Abstract] [Purchase
Article]
An Efficient Support Vector Machine Approach
for Identifying Protein S-Nitrosylation Sites Pp.
573-587
Yu-Xin Li, Yuan-Hai Shao and Ling Jing and Nai-Yang
Deng
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Article]
Targeting Insulin Amyloid Assembly by Aminosugars
and Their Derivatives Pp. 588-593
V. Nagaveni, M. Sravani, S. Prabhakar, B. Sreedhar and
M. Vairamani
[Abstract] [Purchase
Article]
An Antifungal Peptide with Antiproliferative
Activity Toward Tumor Cells from Red Kidney Beans
Pp. 594-600
Miao Li, Hexiang Wang and Tzi Bun Ng
[Abstract] [Purchase
Article]
Expression, Purification and Characterization of the
Escherichia coli Integral Membrane Protein YajC
Pp. 601-608
Jun Fang and Yinan Wei
[Abstract] [Purchase
Article]
Using Random Forest Algorithm to Predict β-Hairpin
Motifs Pp. 609-617
Shao-Chun Jia and Xiu-Zhen Hu
[Abstract] [Purchase
Article]
Isolation and Compositional Analysis
of a CP12-Associated Complex of Calvin Cycle Enzymes from
Nicotiana tabacum Pp. 618-624
A. Elizabete Carmo-Silva, Lucia Marri, Francesca Sparla
and Michael E. Salvucci
[Abstract] [Purchase
Article]
Prediction of Rat Protein Subcellular Localization
with Pseudo Amino Acid Composition Based on Multiple Sequential
Features Pp. 625-633
Ruijia Shi and Cunshuan Xu
[Abstract] [Purchase
Article]
Structural Rationale for the Recognition of Arginine
at P3 in PEXEL Motif Containing
Proteins of Plasmodium falciparum by Plasmepsin V
Pp. 634-641
Lalitha Guruprasad, Karunakar Tanneeru and Kunchur
Guruprasad
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Article]
Novel High-affinity Aβ-binding
Peptides Identified by an Advanced In Vitro Evolution,
Progressive Library Method Pp. 642-650
Sachika Tsuji-Ueno, Masayuki Komatsu, Kakeru Iguchi,
Masahiro Takahashi, Syuhei Yoshino, Miho Suzuki, Naoto Nemoto
and Koichi Nishigaki
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Article]
Abstracts
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Study of Prolactin Permeation Through
the Pericardium and Its Bioavailability Pp. 540-543
Barbara Dolinska, Artur Caban, Lucyna Leszczynska, Grzegorz
Oczkowicz and Florian Ryszka
The prolactin (PRL) permeation through the pericardium depending
on the species of origin (porcine, bovine and ovine) was studied,
and the parameters of its bioavailability were calculated.
An in vitro model using pericardium as a natural
membrane and Frantz cell method was applied.
Significant differences in permeation were observed depending
on the species of origin. Within 5 h, 17.5% of bovine PRL,
27.2% of porcine PRL and 90.3% of ovine PRL permeated the
pericardium. The amount of permeated ovine PRL was 3.3-fold
higher than porcine PRL and 5.2-fold higher than bovine PRL.
The maximum concentration of permeated PRL was reached in
the thirtieth minute of the experiment and was the highest
for ovine PRL (Cmax = 677.21
µg/cm2) and the lowest for
bovine PRL (Cmax=259.97 µg/cm2).
Bioavailability of PRL through the pericardium is 3.3-fold
greater for ovine PRL in comparison to porcine or bovine PRL.
The relative extent of bioavailability for bovine and ovine
prolactin versus the porcine PRL standard was 85.6% and 229.3%,
respectively.
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The Effect of Deleting a Putative Salt Bridge on the
Properties of the Thermostable Subtilisin-Like Proteinase,
Aqualysin I
Jóhanna Arnórsdóttir, Manuela Magnúsdóttir, Ólafur H.
Friðjónsson and Magnús M. Kristjánsson
Aqualysin I, is a subtilisin-like serine proteinase,
from the thermophilic bacterium Thermus aquaticus.
It is predicted that the enzyme contains a salt bridge, D17-R259,
connecting the N- and C-terminal regions of the enzyme. Previously
we reported on the stabilizing effect of the incorporation
of a salt bridge at a corresponding site in VPR, a related
cold adapted enzyme from a marine Vibrio sp. Here
we describe the effect of the reverse change, i.e. the elimination
of the salt bridge on the thermal stability and kinetic properties
of aqualysin I. Deletion of the putative salt bridge in the
D17N mutant of the enzyme destabilized the enzyme by 8-9 °C
in terms of T50%, determined
by thermal inactivation and over 4°C in Tm,
as measured from melting curves of the inhibited enzyme. The
mutation, however, had no significant effect on the kinetic
parameters of the enzyme under standard assay conditions
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Using Pseudo Amino Acid Composition to Predict
Protease Families by Incorporating a Series of Protein Biological
Features
Lele Hu, Lulu Zheng, Zhiwen Wang, Bing Li and
Lei Liu
Proteases are essential to most biological processes
though they themselves remain intact during the processes.
In this research, a computational approach was developed for
predicting the families of proteases based on their sequences.
According to the concept of pseudo amino acid composition,
in order to catch the essential patterns for the sequences
of proteases, the sample of a protein was formulated by a
series of its biological features. There were a total of 132
biological features, which were sourced from various biochemical
and physicochemical properties of the constituent amino acids.
The importance of these features to the prediction is rated
by Maximum Relevance Minimum Redundancy algorithm and then
the Incremental Feature Selection was applied to select an
optimal feature set, which was used to construct a predictor
through the nearest neighbor algorithm. As a demonstration,
the overall success rate by the jackknife test in identifying
proteases among their seven families was 92.74%. It was revealed
by further analysis on the optimal feature set that the secondary
structure and amino acid composition play the key roles for
the classification, which is quite consistent with some previous
findings. The promising results imply that the predictor as
presented in this paper may become a useful tool for studying
proteases.
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Computer-Based Comparison of Structural Features
of Envelope Protein of Alkhurma Hemorrhagic Fever Virus with
the Homologous Proteins of Two Closest Viruses
Hassan Mohabatkar
The aim of this study was prediction of epitopes and
medically important structural properties of protein E of
Alkhurma hemorrhagic fever virus (AHFV) and comparing these
features with two closely relates viruses, i.e. Kyasanur Forest
disease virus (KFDV) and Tick-borne encephalitis virus (TBEV)
by bioinformatics tools. Prediction of evolutionary distance,
localization, sequence of signal peptides, C, N O glycosylation
sites, transmembrane helices (TMHs), cysteine bond positions
and B cell and T cell epitopes of E proteins were performed.
2D-MH, Virus-PLoc, Signal-CF, EnsembleGly, MemBrain, DiANNA,
BCPREDS and MHCPred servers were applied for the prediction.
According to the results, the evolutionary distance of E protein
of AHFV and two other viruses was almost equal. In all three
proteins of study, residues 1-35 were predicted as signal
sequences and one asparagine was predicted to be glycosylated.
Results of prediction of transmembrane helices showed one
TMH at position 444-467 and the other one at position 476-490.
Twelve cysteines were potentially involved to form six disulfide
bridges in the proteins. Four parts were predicted as B cell
epitopes in E protein of AHFV. One epitope was conserved between
three proteins of study. The only conserved major histocompatibility
complex (MHC) binding epitope between three viruses was for
DRB0401 allele. As there are not much experimental data available
about AHFV, computer-aided study and comparison of E protein
of this virus with two closely related flaviviruses can help
in better understanding of medical properties of the virus.
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A Novel Rhodobacter sphaeroides Expression
System for Real-Time Evaluation of Heterologous Protein Expression
Levels
Zhiping Zhao, Zongli Hu, Xin Nie, Lijing Cheng, Guolin
Ding, Min Luo, Yu Pan, Yan Liang and Guoping Chen
Heterologous protein expression levels can not be evaluated
in real time by experimental procedures commonly used for
most expression systems during host cell culture. Rb.
sphaeroides has provided an ideal system for studying
both photosynthesis and membrane development and exhibited
potential as a novel expression system. We constructed the
puc1BA and puc2BA mutant strain Rb.
sphaeroides CQU68 and used it as a novel expression system
to heterologously express proteins fused to β-subunit
of light-harvesting 2 complexes (LH2). The presence of LH2
with β-subunit
fusion proteins was spectrally detected by the LH2 typical
absorption at ~800 nm and ~850 nm, and the formation of these
com-plexes were further confirmed by SDS-PAGE and Western
blot analysis. The expression levels of heterologous protein
measured by SDS-PAGE and Western blot turned out to be higher
as the typical spectral peak heights increase. These findings
suggested that the production of the heterologous protein
could be rapidly detected through the LH2 absorption at ~800
nm and ~850 nm. Moreover, the typical absorption could be
used as a monitor for rapid and real-time evaluation of heterologous
protein expression levels.
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An Efficient Support Vector Machine Approach
for Identifying Protein S-Nitrosylation Sites
Yu-Xin Li, Yuan-Hai Shao and Ling Jing and Nai-Yang
Deng
Protein S-nitrosylation plays a key and specific role
in many cellular processes. Detecting possible S-nitrosylated
substrates and their corresponding exact sites is crucial
for studying the mechanisms of these biological processes.
Comparing with the expensive and time-consuming biochemical
experiments, the computational methods are attracting considerable
attention due to their convenience and fast speed. Although
some computational models have been developed to predict S-nitrosylation
sites, their accuracy is still low. In this work,we incorporate
support vector machine to predict protein S-nitrosylation
sites. After a careful evaluation of six encoding schemes,
we propose a new efficient predictor, CPR-SNO, using the coupling
patterns based encoding scheme. The performance of our CPR-SNO
is measured with the area under the ROC curve (AUC) of 0.8289
in 10-fold cross validation experiments, which is significantly
better than the existing best method GPS-SNO 1.0's 0.685 performance.
In further annotating large-scale potential S-nitrosylated
substrates, CPR-SNO also presents an encouraging predictive
performance. These results indicate that CPR-SNO can be used
as a competitive protein S-nitrosylation sites predictor to
the biological community. Our CPR-SNO has been implemented
as a web server and is available at http://math.cau.edu.cn/CPR
-SNO/CPR-SNO.html.
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Targeting Insulin Amyloid Assembly by Aminosugars
and Their Derivatives
V. Nagaveni, M. Sravani, S. Prabhakar, B. Sreedhar and
M. Vairamani
The use of small carbohydrates that stabilize proteins from
misfolding is important from pharmaceutical point of view.
We have investigated the role of small isomeric amino sugars
on the in vitro aggregation of insulin amyloid. Using
mass spectrometry, we screened 6 isomeric aminosugars for
their role on inhibition of insulin amyloid formation and
the results were compared with transmission electron microscopy
imaging. We found that three N-acetylamino sugars promote
insulin fibril formation. Among three isomeric aminosugars
studied, only galactosamine showed few fibrils whereas other
two isomers showed enhanced fibrils. The results demonstrated
here may contribute to future designing of small amine derivatised
galactose sugars as amyloid inhibitors and understanding their
action.
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An Antifungal Peptide with Antiproliferative
Activity Toward Tumor Cells from Red Kidney Beans
Miao Li, Hexiang Wang and Tzi Bun Ng
A 7.3-kDa antifungal peptide was purified from dried
red kidney beans. The purification procedure entailed ion
exchange chromatography on DEAE-cellulose, affinity chromatography
on Affi-gel blue gel, ion exchange chromatography on CM-cellulose,
followed by fast protein liquid chromatography-gel filtration
on Superdex 75. The peptide was unadsorbed on DEAE-cellulose
but adsorbed on Affi-gel blue gel and CM-cellulose. It exhibited
a molecular mass of 7.3 kDa in gel filtration and also in
SDS-polyacrylamide gel electrophoresis, indicating that it
is a single-chained protein. The N-terminal sequence of the
peptide was DGVCFGGLANGDRT. The peptide exerted an antifungal
action on Fusarium oxysporum with an IC50
of 3.8±0.4 μM
(mean±SD, n=3). It also inhibited mycelial growth in
Mycosphaerella arachidicola. It suppressed growth
of lymphoma MBL2 cells and leukemia L1210 cells with an IC50
of 5.2±0.4 μM
and 7.6±0.6 μM,
respectively. HIV-1 reverse transcriptase was inhibited with
an IC50 of 40±3.2
μM.
However, no activity was demonstrated toward other viral enzymes.
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Expression, Purification and Characterization of the
Escherichia coli Integral Membrane Protein YajC
Jun Fang and Yinan Wei
Escherichia coli YajC is a small integral membrane protein
with a single transmembrane helix. The gene yajC
is part of the secD operon and the protein is identified
in the SecDF-YajC complex. However, the exact function of
YajC remains a mystery. While its function is usually discussed
in the context of the SecDF-YajC complex, studies have shown
that SecD/F, rather than YajC, are essential for those functions.
Recently YajC is identified as the mysterious protein that
co-crystallized with AcrB. To further investigate the structure
of YajC, we expressed and purified the protein in a detergent
solubilized state. The protein assumed a folded structure
containing mixed α/β
secondary structures, consistent with the structural prediction.
Using signal Cys mutations and thiol-specific probes, we found
the C-terminus of YajC was cy-toplasmic, while the N-terminus
of YajC was buried in the membrane. In addition, we expressed
and purified a truncated fragment of YajC that corresponded
to the C-terminal cytoplasmic domain (YajCCT). YajCCT formed
a compact structure rich in β-strands
and existed as a trimer.
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Using Random Forest Algorithm to Predict β-Hairpin
Motifs
Shao-Chun Jia and Xiu-Zhen Hu
A novel method is presented for predicting β-hairpin
motifs in protein sequences. That is Random Forest algorithm
on the basis of the multi-characteristic parameters, which
include amino acids component of position, hydropathy component
of position, predicted secondary structure information and
value of auto-correlation function. Firstly, the method is
trained and tested on a set of 8,291 β-hairpin
motifs and 6,865 non-β-hairpin
motifs. The overall accuracy and Matthew’s correlation
coefficient achieve 82.2% and 0.64 using 5-fold cross-validation,
while they achieve 81.7%and 0.63 using the independent test.
Secondly, the method is also tested on a set of 4,884 β-hairpin
motifs and 4,310 non-β-hairpin
motifs which is used in previous studies. The overall accuracy
and Matthew’s correlation coefficient achieve 80.9%
and 0.61 for 5-fold cross-validation, while they achieve 80.6%
and 0.60 for the independent test. Compared with the previous,
the present result is better. Thirdly, 4,884 β-hairpin
motifs and 4,310 non-β-hairpin
motifs selected as the training set, and 8,291 β-hairpin
motifs and 6,865 non-β-hairpin
motifs selected as the independent testing set, the overall
accuracy and Matthew’s correlation coefficient achieve
81.5% and 0.63 with the independent test.
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Isolation and Compositional Analysis
of a CP12-Associated Complex of Calvin Cycle Enzymes from
Nicotiana tabacum
A. Elizabete Carmo-Silva, Lucia Marri, Francesca Sparla
and Michael E. Salvucci
Two Calvin Cycle enzymes, NAD(P)-dependent glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a
multiprotein complex with CP12, a small intrinsically-unstructured
protein. Under oxidizing conditions, association with CP12
confers redox-sensitivity to the otherwise redox-insensitive
A isoform of GAPDH (GapA) and provides an additional level
of down-regulation to the redox-regulated PRK. To determine
if CP12-mediated regulation is specific for GAPDH and PRK
in vivo, a high molecular weight complex containing CP12
was iso-lated from tobacco chloroplasts and leaves and its
protein composition was characterized. Gel electrophoresis
and immu-noblot analyses after separation of stromal proteins
by size fractionation verified that the GAPDH (both isoforms)
and PRK co-migrated with CP12 in dark- but not light-adapted
chloroplasts. Nano-liquid-chromatography-mass-spectrometry
of the isolated complex identified only CP12, GAPDH and PRK.
Since nearly all of the CP12 from darkened chloroplasts migrates
with GADPH and PRK as a high molecular mass species, these
data indicate that the tight association of tobacco CP12 with
GAPDH and PRK is specific and involves no other Calvin Cycle
enzymes.
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Prediction of Rat Protein Subcellular Localization
with Pseudo Amino Acid Composition Based on Multiple Sequential
Features
Ruijia Shi and Cunshuan Xu
The study of rat proteins is an indispensable task in
experimental medicine and drug development. The function of
a rat protein is closely related to its subcellular location.
Based on the above concept, we construct the benchmark rat
proteins dataset and develop a combined approach for predicting
the subcellular localization of rat proteins. From protein
primary sequence, the multiple sequential features are obtained
by using of discrete Fourier analysis, position conservation
scoring function and increment of diversity, and these sequential
features are selected as input parameters of the support vector
machine. By the jackknife test, the overall success rate of
prediction is 95.6% on the rat proteins dataset. Our method
are performed on the apoptosis proteins dataset and the Gram-negative
bacterial proteins dataset with the jackknife test, the overall
success rates are 89.9% and 96.4%, respectively. The above
results indicate that our proposed method is quite promising
and may play a complementary role to the existing predictors
in this area.
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Structural Rationale for the Recognition of Arginine
at P3 in PEXEL Motif Containing
Proteins of Plasmodium falciparum by Plasmepsin V
Lalitha Guruprasad, Karunakar Tanneeru and Kunchur
Guruprasad
The virulent form of malaria is caused by Plasmodium
falciparum that infects red blood cells. In order to
survive inside the host, the parasite remodels the infected
erythrocytes by exporting more than 300 effector proteins
outside the parasitophorous vacuole membrane into the cytosol.
The main feature of all the export proteins is the presence
of a pentapeptide sequence motif; RxLxE/Q/D. This sequence
motif is hydrolysed between L – x and the proteins with
the ace-tylated new N-terminus xE/Q/D are exported. The enzyme
responsible for this hydrolysis is plasmepsin V which is one
of the ten aspartic proteases in P. falciparum. In
order to understand the structural rationale for the specificity
of this protease towards cleavage of the above motif, we generated
three-dimensional models of seven plasmepsins (I, V to X)
for which experimental structures are not available and compared
these along with the crystal structures of three P. falciparum
plasmepsins (II to IV). The structure comparisons revealed
the importance of Tyr13, Glu77 and Ala117 specific to plas-mepsin
V that facilitates the accommodation of arginine at P3
in the RxLxE/Q/D motif. Our analysis correlates the structure-function
relationship of plasmepsin V.
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Novel High-affinity Aβ-binding
Peptides Identified by an Advanced In Vitro Evolution,
Progressive Library Method
Sachika Tsuji-Ueno, Masayuki Komatsu, Kakeru Iguchi,
Masahiro Takahashi, Syuhei Yoshino, Miho Suzuki, Naoto Nemoto
and Koichi Nishigaki
Recent studies have been supporting that the generation
of Aβ42
oligomers is responsible for Alzheimer’s disease. Therefore,
those peptides which bind to Aβ42
are scientifically interesting and can be possible candidates
for the diagnosis and therapy of Alzheimer’s disease.
A systemic in vitro evolution, developed recently
and the designated progressive library method (PLM), was applied
to obtain Aβ42-binding
aptamers peptides. As a result, high affinity peptide aptamers
made of 8 or 9 amino acids could be identified by this approach,
endorsing the methodological effectiveness. Namely, the selection
products from the secondary library of diversified peptides,
which was constructed based on the information obtained from
the primary library selection, were confirmed to be superior
to those selected from the primary library as had been reported
previously. The affinities of those peptides measured by SPR
(surface plasmon resonance) were comparable to or higher than
that of those peptides so far reported (Kd
of 10-7). The other peptides
selected were confirmed of their binding by a novel mode of
gel shift assay (fluorescence enhancement caused by the binding).
Thus, novel Aβ42-binding
peptides with high affinity were provided for the future Alzheimer’s
disease study. The demonstration of the effectiveness of the
systemic in vitro evolution of PLM is very encouraging
for the study of identifying novel functional peptides.
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