| Protein
& Peptide Letters
ISSN: 0929-8665
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Articles
Crystallization and Preliminary Crystallographic
Analysis of Laminarinase from Rhodothermus marinus:
A Case of Pseudomerohedral Twinning
Alexander M. Golubev,
Adriana L. Rojas,
Alessandro S. Nascimento,
Lucas Bleicher, Anna A.
Kulminskaya, Elena V. Eneyskaya
and Igor Polikarpov
[Abstract]
Structure-Function Study of a Plasmodium
falciparum Hsp70 Using Three Dimensional Modelling and
In Vitro Analyses
Addmore Shonhai
Melissa Botha, Tjaart
A. P. de Beer, Aileen Boshoff and Gregory L. Blatch
[Abstract]
Function Prediction of Hypothetical Proteins
without Sequence Similarity to Proteins of Known Function
S. Kannan, A.M. Hauth and G. Burger
[Abstract]
Crystallization and Preliminary X-Ray Crystallographic
Analysis of Human Plasma Platelet Activating Factor Acetylhydrolase
Uttamkumar Samanta,
Cheryl Wilder and Brian
J. Bahnson
[Abstract]
Substrate Specificity of Rat DESC4, A Type II
Transmembrane Serine Protease
Maik Behrens,
Friedrich Buck and
Wolfgang Meyerhof
[Abstract]
Identification of a Biosurfactant Producing Strain:
Bacillus subtilis HOB2
Namir I.A. Haddad, Ji Wang and Bozhong Mu
[Abstract]
Mechanisms of Prion Protein Aggregation
Sarah N. Fontaine and David R. Brown
[Abstract]
Molecular Cloning and Expression of Several New
Anopheles cracens Epsilon Class Glutathione Transferases
Jeerang Wongtrakul,
Jantana Wongsantichon,
Ardcharaporn Vararattanavech,
Posri Leelapat, La-aied
Prapanthadara and Albert
J. Ketterman
[Abstract]
Pomegranin, An Antifungal Peptide from Pomegranate
Peels
Guang Guoa, He Xiang Wang
and Tzi Bun Ng
[Abstract]
Design, Synthesis and Docking Studies of Hydroxyethylamine
and Hydroxyethylsulfide BACE-1 Inhibitors
Luca Rizzi, Nadia Vaiana,
Francesca Sagui, Eva Genesio,
Elena Pilli,Valentina Porcari
and Sergio Romeo
[Abstract]
Proteinase K-Resistant Aggregates of Recombinant Prion Protein
PrP-(23-98) are Toxic to Cultured Cells
Noriyuki Shiraishi, Yoko
Inai and Yoshito Ihara
[Abstract]
Prediction of Protein Secondary Structure Content
by Using the Concept of Chou’s Pseudo Amino Acid Composition
and Support Vector Machine
Chen, Lixuan Chen,
Xiaoyong Zou and Peixiang
Cai
[Abstract]
Hydroxyl Radical Mediates Oxidative Modification
of Caprine Alpha-2 Macroglobulin
Shakil A. Khan
and Fahim H. Khan
[Abstract]
The Inhibitory Effect of Propofol on Bovine Lactoperoxidase
Melda Sisecioglu, Murat Çankaya, Ilhami Gülçin
and Hasan Özdemir
[Abstract]
Correlation Between Protein Sequence Similarity
and X-Ray Diffraction Quality in the Protein Data Bank
Hui-Meng Lu, Da-Chuan Yin,
Ya-Jing Ye, Hui-Min Luo, Li-Qiang Geng, Hai-Sheng Li, Wei-Hong
Guo and Peng Shang
[Abstract]
Crystallization and Preliminary Diffraction Studies
of Nudix Hydrolase YmfB from Escherichia coli K-1
Myoung-Ki Hong,
Jin-Kwang Kim, Yeh-Jin Ahn
and Lin-Woo Kang
[Abstract]
Structure Solution of Misfolded Conformations
Adopted by Intrinsically Disordered Alzheimer's Tau Protein
Jozef Sevcik,
Rostislav Skrabana, Eva
Kontsekova and Michal
Novak
[Abstract]
Expression of HPV 58 Long and Short L1 Capsid
Proteins in Primary Mouse Keratinocyte Cultures
Xiao Wang,
Juan Liu, Wei Ming Zhao
and Kong-Nan Zhao
[Abstract]
Abstracts
[Back to top]
Crystallization and Preliminary Crystallographic Analysis
of Laminarinase from Rhodothermus marinus: A Case
of Pseudomerohedral Twinning
Alexander M. Golubev,
Adriana L. Rojas,
Alessandro S. Nascimento,
Lucas Bleicher, Anna A.
Kulminskaya, Elena V. Eneyskaya
and Igor Polikarpov
Thermophilic endo-1,3(4) β
glucanase (laminarinase) from Rhodothermus marinus
was crystallized by the hanging-drop vapor diffusion method.
The needle-like crystals belong to space group P21
and contain two protein molecules in the asymmetric unit with
a solvent content of 51.75 %. Diffraction data were collected
to a resolution of 1.95Å
and resulted in a dataset with an overall Rmerge
of 10.4% and a completeness of 97.8%. Analysis of the structure
factors revealed pseudomerohedral twinning of the crystals
with a twin fraction of approximately 42%.
[Back to top]
Structure-Function Study of a Plasmodium
falciparum Hsp70 Using Three Dimensional Modelling and
In Vitro Analyses
Addmore Shonhai
Melissa Botha, Tjaart
A. P. de Beer, Aileen Boshoff and Gregory L. Blatch
The spatial orientation of domains of the heat shock
protein 70 from Plasmodium falciparum (PfHsp70) were
mapped based on a three-dimensional model of the protein.
Purified PfHsp70 displayed chaperone activity in vitro.
Amino acid substitutions introduced in the chaperone’s
substrate binding cavity compromised the protein’s chaperone
function.
[Back to top]
Function Prediction of Hypothetical Proteins
without Sequence Similarity to Proteins of Known Function
S. Kannan, A.M. Hauth and G. Burger
Function prediction by sequence-similarity based methods
identifies only ~50% of the proteins deduced from newly sequenced
genomes. We have developed an approach to annotate the ‘leftover
proteins’ i.e., those which cannot be assigned function
using sequence similarity. Our method (MOPS) is pan-taxonomic,
predicting fine-grained molecular function (rather than a
broad functional category) with high performance. In addition,
we developed a validation scheme that assesses predictions
using domain-specific knowledge.
[Back to top]
Crystallization and Preliminary X-Ray Crystallographic
Analysis of Human Plasma Platelet Activating Factor Acetylhydrolase
Uttamkumar Samanta,
Cheryl Wilder and Brian J. Bahnson
The plasma form of the human enzyme platelet activating
factor acetylhydrolase (PAF-AH) has been crystallized, and
X-ray diffraction data were collected at a synchrotron source
to a resolution of 1.47 Å. The crystals belong to space
group C2, with unit cell parameters of α
= 116.18, b = 83.06, c = 96.71 Å,
and β=
115.09° and two molecules in the asymmetric unit. PAF-AH
functions as a general anti-inflammatory scavenger by reducing
the levels of the signaling molecule PAF. Additionally, the
LDL bound enzyme has been linked to atherosclerosis due to
its hydrolytic activities of pro-inflammatory agents, such
as sn-2 oxidatively fragmented phospholipids.
[Back to top]
Substrate Specificity of Rat DESC4, A Type II
Transmembrane Serine Protease
Maik Behrens,
Friedrich Buck and
Wolfgang Meyerhof
Type II transmembrane serine proteases (TTSPs) are involved
in important physiological processes, such as pro-hormone
processing, cellular signaling, host immune defense, and cancer
development. The diversity of functions is reflected by the
multidomain architecture of these proteases, which are composed
of a variety of functional domains in addition to the catalytic
domain. Recently, we identified rat DESC4, a member of the
HAT/DESC1-like subfamily of TTSPs. Intriguingly, DESC4 gene
expression is confined to few tissues including gustatory
papillae. In the current publication we present the purification
of the catalytic domain of recombinant rat DESC4. Subsequently,
the catalytic domain was subjected to a refolding procedure.
During refolding we observed endogenous catalytic activity
leading to smaller fragments, which were analyzed by peptide
sequencing. The identified cleavage-sites are typical for
trypsin-like serine proteases. For further analyses a homology-based
model of the DESC4 catalytic domain was generated enabling
us to investigate protease-substrate interaction in more detail.
[Back to top]
Identification of a Biosurfactant Producing Strain:
Bacillus subtilis HOB2
Namir I.A. Haddad, Ji Wang and Bozhong Mu
A biosurfactant-producing strain was isolated from the
production water of an oil-field and was identified as Bacillus
subtilis HOB2 by 16S rRNA gene sequencing. The production
of biosurfactant by Bacillus subtilis HOB2 has been
investigated using different carbon and nitrogen sources,
under thermophilic and mesophilic conditions. The strain was
able to grow and to produce surfactant, reducing the surface
tension of medium to 27 mN/m on sucrose, and 28 mN/m on glucose
after 24 h of cultivation. The strain was able to produce
the maximum amount of biosurfactant when ammonium ions were
used as nitrogen source. The surface-active compound was stable
during exposure to elevate temperature (100oC), high salinity
(25% NaCl) and a wide range of pH values (5.0-11.0). The biosurfactant
was capable of forming a promising emulsification index (E24=
68%) with kerosene. The kinetic studies revealed that biosurfactant
production is a cell growth-associated process. Preliminary
chemical characterization revealed that the surfactant has
a lipopeptide composition similar to surfactin as confirmed
by TLC and IR analysis. Properties and characteristics of
the biosurfactant produced by Bacillus subtilis HOB2
suggesting potential commercial applications, such as enhanced
oil recovery, bioremediation of soil and marine environments,
and food industries.
[Back to top]
Mechanisms of Prion Protein Aggregation
Sarah N. Fontaine and David R. Brown
The prion protein is a cell surface glycoprotein that
is converted to a protease resistant abnormal isoform during
the course of prion disease. The normal isoform of this protein
has been shown to be an antioxidant that aids the survival
of neurones. The abnormal isoform is associated with both
the transmissible agent of prion diseases and is also toxic.
Recent studies have shown that there are multiple end states
in terms of aggregation of the protein. Both soluble oligomers
and insoluble fibrils can form from the abnormal isoform.
Although fibrils are characteristic of the disease, the most
infectious prions are associated with oligomers. Neurotoxicity
can be associated with fibrils but mostly appears to be due
to small aggregates. For many years fibrils were believed
to be central to the disease process but currently evidence
supports the notion that fibrils represent a "bulk"
form of abnormal protein, which is largely inert, but carried
along a small active component. This review will examine what
is known about the mechanisms behind prion protein aggregation,
and the relevance of each form for the disease.
[Back to top]
Molecular Cloning and Expression of Several New Anopheles
cracens Epsilon Class Glutathione Transferases
Jeerang Wongtrakul,
Jantana Wongsantichon,
Ardcharaporn Vararattanavech,
Posri Leelapat, La-aied
Prapanthadara and Albert
J. Ketterman
Glutathione transferases, GSTs, are detoxification proteins
that are found in most organisms. The acGSTE3-3 had the ability
to conjugate 4-hydroxynonenal, a cytotoxic lipid peroxidation
product. Although other Epsilon GSTs showed roles in insecticide
metabolism, the acGSTE3-3 appeared to have a major role in
detoxifying lipid peroxidation products conferring protection
against oxidative damage.
[Back to top]
Pomegranin, An Antifungal Peptide from Pomegranate
Peels
Guang Guoa, He Xiang Wang
and Tzi Bun Ng
A new antifungal peptide designated as pomegranin, with an
N-terminal sequence resembling that of rice disease resistance
NB-S-LRR-like protein, was isolated from fresh pomegranate
peels by ion exchange chromatography on DEAE-cellulose, affinity
chromatography on Affi-gel blue gel, and gel filtration by
fast protein liquid chromatography on Superdex 75. Pomegranin
was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel
blue gel. It exhibited a molecular mass of 11 kDa in both
gel filtration and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. It inhibited mycelial growth in the fungi
Botrytis cinerea and Fusarium oxysporum
with an IC50 of 2 µM and 6.1 µM,
respectively. It was devoid of hemagglutinating, ribonuclease,
deoxyribonuclease and protease inhibitory activities.
[Back to top]
Design, Synthesis and Docking Studies of Hydroxyethylamine
and Hydroxyethylsulfide BACE-1 Inhibitors
Luca Rizzi,
Nadia Vaiana, Francesca
Sagui, Eva Genesio,
Elena Pilli,Valentina Porcari
and Sergio Romeo
Both stereoisomer of hydroxyethylamine (HEA) and hydroxyethylsulfide
(HES) transition-state isostere inhibitors of BACE-1 were
synthesized. The syn-HEA epimer resulted always more active
than the anti stereoisomer independently from the P1
and the P1’ substituents. On the contrary,
the anti epimer of the HES isostere resulted more active than
the syn stereoisomer. The change of stereopreference was studied
by molecular modelling.
[Back to top]
Proteinase K-Resistant Aggregates of Recombinant Prion
Protein PrP-(23-98) are Toxic to Cultured Cells
Noriyuki Shiraishi, Yoko
Inai and Yoshito Ihara
Here, we show for the first time that non-fibrillar and spherical
aggregates produced from PrP-(23-98) in the presence of NADPH
plus copper ions are toxic to cultured cells and induce apoptotic
signals. It is also confirmed that endogenous cellular PrP
isoform is not required for toxicity to occur.
[Back to top]
Prediction of Protein Secondary Structure Content
by Using the Concept of Chou’s Pseudo Amino Acid Composition
and Support Vector Machine
Chen, Lixuan Chen,
Xiaoyong Zou and Peixiang
Cai
Protein secondary structure carries information about
local structural arrangements. Significant majority of successful
methods for predicting the secondary structure is based on
multiple sequence alignment. However, the multiple alignment
fails to achieve accurate results when a protein sequence
is characterized by low homology. To this end, we propose
a novel method for prediction of secondary structure content
through comprehensive sequence representation. The method
is featured by employing a support vector machine (SVM) regressing
system and adopting a different pseudo amino acid composition
(PseAAC), which can partially take into account the sequence-order
effects to represent protein samples. It was shown by both
the self-consistency test and the independent-dataset test
that the trained SVM has remarkable power in grasping the
relationship between the PseAAC and the content of protein
secondary structural elements, including α-helix, 310-helix,
π-helix,
β-strand, β-bridge, turn, bend and the rest
random coil. Results prior to or competitive with the popular
methods have been obtained, which indicate that the present
method may at least serve as an alternative to the existing
predictors in this area.
[Back to top]
Hydroxyl Radical Mediates Oxidative Modification of Caprine
Alpha-2 Macroglobulin
Shakil A. Khan
and Fahim H. Khan
ROS, including .OH, is now recognized as the hallmark
of inflammation and damage to the anti-proteinase barrier
has been implicated in a number of pathophysiological conditions.
We have previously shown that O2.-
and H2O2/HOCl
are physiologically relevant inactivators of α2M,
a key anti-proteinase. Here, we show that .OH disrupted caprine
α2M structure and
antiproteolytic potential in vitro, suggesting that
its function could be compromised via oxidative modification.
[Back to top]
The Inhibitory Effect of Propofol on Bovine Lactoperoxidase
Melda Sisecioglu, Murat Çankaya, Ilhami
Gülçin and Hasan
Özdemir
Propofol (2,6-diisopropylphenol) is a hypnotic intravenous
agent with in vivo antioxidant properties. This study
was undertaken to examine the in vitro effect of
propofol on lactoperoxidase (LPO; E.C. 1.11.1.7) obtained
from bovine milk. Lactoperoxidase was purified with three
purification steps: Amberlite CG-50 resin, CM-Sephadex C-50
ion-exchange chromatography and Sephadex G-100 gel filtration
chromatography, respectively. Lactoperoxidase was purified
with a yield of 21.6%, a specific activity of 34 EU/mg proteins
and 14.7-fold purification. One enzyme unit is defined as
the oxidation of 1 µmol ABTS per min under the assay
condition (25oC, pH: 6.0).
To determine enzyme purity, sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) was performed and single band
was observed. The effect of propofol on lactoperoxidase were
determined using 2,2'-azino-bis (3-ethylbenzthiazoline-6 sulfonic
acid) diammonium salt (ABTS) as a chromogenic substrate. The
IC50 value of propofol was
found as 15.97 µM. Also, Ki
constant for propofol was 3.72 µM and propofol was found
as competitive inhibitor.
[Back to top]
Correlation Between Protein Sequence Similarity
and X-Ray Diffraction Quality in the Protein Data Bank
Hui-Meng Lu, Da-Chuan Yin,
Ya-Jing Ye, Hui-Min Luo, Li-Qiang Geng, Hai-Sheng Li, Wei-Hong
Guo and Peng Shang
As the most widely utilized technique to determine the 3-dimensional
structure of protein molecules, X-ray crystallography can
provide structure of the highest resolution among the developed
techniques. The resolution obtained via X-ray crystallography
is known to be influenced by many factors, such as the crystal
quality, diffraction techniques, and X-ray sources, etc.
In this paper, the authors found that the protein sequence
could also be one of the factors. We extracted information
of the resolution and the sequence of proteins from the Protein
Data Bank (PDB), classified the proteins into different clusters
according to the sequence similarity, and statistically analyzed
the relationship between the sequence similarity and the best
resolution obtained. The results showed that there was a pronounced
correlation between the sequence similarity and the obtained
resolution. These results indicate that protein structure
itself is one variable that may affect resolution when X-ray
crystallography is used.
[Back to top]
Crystallization and Preliminary
Diffraction Studies of Nudix Hydrolase YmfB from Escherichia
coli K-1
Myoung-Ki Hong,
Jin-Kwang Kim, Yeh-Jin Ahn
and Lin-Woo Kang
Nudix hydrolases are a family of proteins that contains
the Nudix signature of the characteristic amino-acid sequence
Gx5Ex5[UA]xREx2EExGU,
where x represents any amino acid and U usually a bulky hydrophobic
amino acid, such as Leu, Val, or Ile. They ubiquitously exist
in more than 200 species. YmfB, a novel Nudix hydrolase found
in Escherichia coli, is a nonspecific nucleoside
tri- and di-phosphatase, which atypically releases inorganic
orthophosphate from triphosphates instead of pyrophosphate.
In this study, YmfB was cloned, overexpressed, and crystallized.
Two different crystals, one belonging to an orthorhombic space
group C2221 and the other
a monoclinic space group P21,
diffracted to 2.7 Å and 2.6 Å resolution, and
had unit cell parameters of α = 68.7 Å,
b = 283.1 Å, c = 70.4 Å and
α = 69.1 Å, b = 70.3 Å,
c = 145.6 Å, β = 103.3o,
respectively. For the C2221
space group, four or five monomers exist in the asymmetric
unit, with a corresponding Vm of 2.48 or 1.99 Å3
Da-1 and a solvent content
of 50.5 or 38.2%. For the P21
space group, eight or nine monomers exist in the asymmetric
unit, with a corresponding Vm
of 2.49 or 2.21 Å3
Da-1 and a solvent content
of 50.7 or 44.5%.
[Back to top]
Structure Solution of
Misfolded Conformations Adopted by Intrinsically Disordered
Alzheimer's Tau Protein
Jozef Sevcik,
Rostislav Skrabana, Eva
Kontsekova and Michal
Novak
Until now it was impossible to obtain atomic structure
of intrinsically disordered protein (IDP) tau and/or its assembly
in Alzheimer’s paired helical filaments as neither of
them could have been prepared in the form amenable to X-ray
or NMR techniques. Using IDP tau property to attain regular
tertiary structure after binding events during self-assembly
or when complexed with its target we propose monoclonal antibodies
as surrogate tau protein binding partners to form complexes
and crystals for structure solution by X-ray technique.
[Back to top]
Expression
of HPV 58 Long and Short L1 Capsid Proteins in Primary Mouse
Keratinocyte Cultures
Xiao Wang,
Juan Liu, Wei Ming Zhao
and Kong-Nan Zhao
We studied expression of HPV 58 long and short L1
proteins in primary mouse keratinocyte (KC) cultures by transient
transfection of the L1 expression constructs. Following transient
transfection, long and short L1 open reading frames (ORFs)
were transcribed continuously for 9 days; however, no significant
difference was detected between the long and short L1 mRNA
levels measured by quantitative RT-PCR. Western blot analysis
showed that both long and short L1 proteins were continuously
detected in L1-transfected KCs for 9 days post-transfection
and the significantly increased signals of the L1 proteins
over time were associated with KC differentiation. Moreover,
L1 protein was more abundant in KCs transfected with the short
L1 ORF than the long L1 ORF. In vitro translation
of the L1 mRNAs indicated further that the short L1 mRNA had
significantly higher translation efficiency than the long
L1 mRNA in cell-free lysate system. The L1 proteins expressed
from the two L1 mRNAs in KCs were similarly stable. Thus,
approximate 40% lower level of expression of the L1 protein
in KCs transfected with the long L1 ORF was probably due to
a stem-loop structure with high ΔG value downstream
the first AUG codon in its mRNA secondary structure. This
stem-loop structure might prevent efficient binding of the
ribosome to mRNA and therefore reduced translation.
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