| Protein
& Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 15, Number 9, 2008
Contents
Special Board Members Issue
Editorial: Pp.
866-867
Targeting the Plasmepsin 4 Orthologs of Plasmodium
sp. with “Double Drug” Inhibitors Pp.
868-873
L. Janka, J. Clemente, N. Vaiana, A. Sparatore,
S. Romeo and B.M. Dunn
[Abstract] [Purchase
Article]
Structural and Biochemical Investigation
of Heptad Repeat Derived Peptides of Human SARS Corona Virus
(hSARS-CoV) Spike Protein Pp. 874-886
S. Basak, X. Hao, A. Chen, M. Chrétien
and A. Basak
[Abstract] [Purchase
Article]
Identification of a Thermo-Regulated
Glutamine-Binding Protein from Yersinia pestis Pp.
887-894
M. Cosman, J.B. Pesavento, A. Zemla, P.T.
Beernink, R. Balhorn and D. Barsky
[Abstract] [Purchase
Article]
Is Asparagine Deamidation in the Porcine
Odorant-Binding Protein Related to the Odor Molecules Binding?
Pp. 895-899
G. Mamone and S. D’Auria
[Abstract] [Purchase
Article]
Pt2L4 Protein, a Homologue to Hev b 5 from Rubber Tree, May
Not Be Responsible for the Cross-Reactions to Cassava Shown
by People Allergic to Latex Pp. 900-902
C.R.B. de Souza, D. Beezhold and
L.J.C.B. Carvalho
[Abstract] [Purchase
Article]
Structural Refinement of Insecticidal
Plant Proteinase Inhibitors from Nicotiana alata Pp.
903-909
H.J. Schirra, M.A. Anderson and D.J.
Craik
[Abstract] [Purchase
Article]
Binding Mode of α-Conotoxins
to an Acetylcholine Binding Protein Determined by Saturation
Transfer Difference NMR Pp. 910-914
J.-C. Westermann, R.J. Clark and
D.J. Craik
[Abstract] [Purchase
Article]
Predicting Membrane Protein Types by
the LLDA Algorithm Pp. 915-921
T. Wang, J. Yang, H.-B. Shen and
K.-C. Chou
[Abstract] [Purchase
Article]
Influenza A Virus M1 Protein Structure
Probed by In Situ Limited Proteolysis with Bromelain
Pp. 922-930
L.V. Kordyukova, M.V. Serebryakova, V.Y.
Polyakov, T.V. Ovchinnikova, Yu. A. Smirnova, N.V. Fedorova
and L.A. Baratova
[Abstract] [Purchase
Article]
Elucidation of Structural Requirements
of Mastoparan for Mast Cell Activation: Toward the Comprehensive
Prediction of Cryptides Acting on Mast Cells Pp.
931-937
H. Mukai, Y. Suzuki, Y. Kiso and
E. Munekata
[Abstract] [Purchase
Article]
Characterization of the Active Site and
a Unique Uncompetitive Inhibitor of the PPM1-Type Protein
Phosphatase PPM1D Pp. 938-948
Y. Chuman, H. Yag, T. Fukuda, T. Nomura,
M. Matsukizono, Y. Shimohigashi and K. Sakaguchi
[Abstract] [Purchase
Article]
Radar Chart Deviation Analysis of Prion
Protein Amino Acid Composition Defines Characteristic Structural
Abnormalities of the N-Terminal Octapeptide
Tandem Repeat Pp. 949-955
S. Yokotani, T. Nose, Y. Horiuchi, A. Matsushima
and Y. Shimohigashi
[Abstract] [Purchase
Article]
TOP-IDP-Scale: A New Amino Acid Scale
Measuring Propensity for Intrinsic Disorder Pp.
956-963
A. Campen, R.M. Williams, C.J. Brown, J.
Meng, V.N. Uversky and A.K. Dunker
[Abstract] [Purchase
Article]
Structural Fragments in Protein Model
Refinement Pp. 964-971
S.M. Hollup, W.R. Taylor and I.
Jonassen
[Abstract] [Purchase
Article]
Equilibrium Folding of Porcine Insulin
Precursor in the Presence of Redox Buffer: Implications for
the Common Intermediates Shared by Its Unfolding/Refolding
Processes Pp. 972-979
J. Zhao, Q.-L. Huang, Y.-H. Tang, Z.-Y.
Guo, Z.-S. Qiao, G.-J. Xu and Y.-M. Feng
[Abstract] [Purchase
Article]
Synthesis and Structural Analysis of
6-Aminobicyclo[2.2.1]heptane-2-carboxylic Acid as a Conformationally
Constrained γ-Turn
Mimic Pp. 980-984
J.-S. Park, K.R. Kim, H.Y. Nam, C.-E. Yeom,
C. Chough, S.H. Kwon, S. Ro, D.-K. Shin and B.M.
Kim
[Abstract] [Purchase
Article]
Strategies for Recombinant Expression
of Small, Highly Disulphide-Bonded, Cationic Antimicrobial
Peptides Pp. 985-994
A.L. Greenshields, L.C. Knickle, R. Syvitski
and S.E. Douglas
[Abstract] [Purchase
Article]
Neutrophil Proteome: Lessons from Different
Standpoints Pp. 995-1001
C.F.M. Morris, M.S. Castro and
W. Fontes
[Abstract] [Purchase
Article]
Biochemical and Structural Investigations
of Bothropstoxin-II, a Myotoxic Asp49 Phospholipase A2
from Bothrops jararacussu Venom Pp.
1002-1008
M.T. Murakami, M.R. Lourenzoni, E.Z. Arruda,
M.A. Tomaz, M.M. Viçoti, J.R.B. Abrego, P.A. Melo and
R.K. Arni
[Abstract] [Purchase
Article]
Purification and Characterization of a Novel Protease
from the Latex of Pedilanthus tithymaloides Pp.
1009-1016
R. Bhowmick, N.K.P. Kumari, M.V. Jagannadham
and A.M. Kayastha
[Abstract] [Purchase
Article]
A Novel Thermoacidophilic Cellulase from
Alicyclobacillus acidocaldarius Pp. 1017-1021
A. Morana, A. Esposito, L. Maurelli, G.
Ruggiero, E. Ionata, M. Rossi and F. La Cara
[Abstract] [Purchase
Article]
Major Digestive Carbohydrase During Larval
Development of Meal Moth, Plodia interpunctella (Lepidoptera:
Pyralidae) Pp. 1022-1026
M.P. de Sales, A. Alcazar, L.M. Lima, T.M.L.
Amorim, J.C.M. Pitanga, R.A. Pereira, L.L.P. Macedo, F.P.
Macedo, A.S. Oliveira and A.F. Uchôa
[Abstract] [Purchase
Article]
Purification and Characterization of
Recombinant Lipid Storage Protein-2 from Drosophila melanogaster
Pp. 1027-1032
E.L. Arrese, L. Rivera, M. Hamada and
J.L. Soulages
[Abstract] [Purchase
Article]
Abstracts
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Editorial:
First a few notes on the operation of the journal:
We have just made the decision to change to 12 issues per
year starting in 2009, up from 10 issues this year. This is
due to the very heavy manuscript flow that we have been experiencing
in 2008. In addition, the larger size and two column format,
initiated in 2005 with volume 12, have been well received.
Improvements have also been made with respect to reproduction
of figures, although this will always be dependent upon getting
high quality files from the authors. We have seen a growth
in the Impact Factor and expect this to continue. I want to
express my appreciation to Miss Sarwat Aziz Abbasi of the
publisher’s office for all her help with all matters
involving the journal.
We are very grateful to the members of the Editorial Advisory
Board (EAB) for Protein and Peptide Letters for their efforts
over the years to review manuscripts, to submit their own
work, to recommend referees, and to critique the operation
of all aspects of the journal. Over the past 15 years we have
added new members to the EAB, thanked retiring members, and
added new Regional Editors to help with the increasing manuscript
flow we have experienced. While we are actively soliciting
new individuals at this time, we are always open to self-nomination
from interested readers of the journal.
In this issue, we inaugurate a special feature that we hope
will prove to be valuable for all readers. This issue features
contributions from members of the EAB. This should help other
scientists to identify with some EAB member when submitting
their work to PPL. By identifying an EAB member who works
in the area that you do to oversee the peer review process,
you can insure that your manuscript will get a fair analysis
by an expert in the field. For the same reason, to insure
fair peer review, we ask that all submissions be accompanied
by a list of four or more potential referees.
The contributions in this issue were subjected to the same
peer review process as all manuscripts and all went through
a round of revision. The contributions cover a wide range
of protein and peptide science. The first five papers loosely
deal with studies of binding interactions between proteins
and other molecules. Dunn and colleagues report on of a series
of statine-based inhibitors binding to enzymes of the plasmepsin
family as part of a study on potential antimalarial drugs.
Ajoy Basak and coworkers report on the properties of heptad
repeats from a fragment of the spike surface glycoprotein
of the SARS virus. Monique Cosman, Daniel Barsky and their
colleagues discuss the function of a specific Yersinia
pestis gene product and conclude that it is most likely
a glutamine binding protein. Odorant binding proteins are
β-barrel
proteins with the cavity inside the barrel serving as the
binding site. Mamone and D’Auria report on the effects
of deamination of a porcine odorant binding protein on its
function. Cláudia Regina Batista de Souza and her colleagues
examine the possible cross reaction between sensitivity to
latex and to proteins from the cassava plant.
The next ten papers deal with structural analysis and prediction.
David Craik’s lab has contributed two papers to this
issue: in the first, Schirra, Anderson, and Craik report on
the structural refinement of NMR analysis of protease inhibitors
from the tobacco plant; in the second paper, Westermann, Clark,
and Craik use NMR to study the binding mode of α-conotoxins
to an acetylcholine binding protein. Predicting membrane protein
types is a new process of great importance in this post-genomic
world. Kuo-Chen Chou and his colleagues apply a new program,
called Local Linear Discriminant Analysis, to this job.
The matrix protein M1 plays an important role in influenza
virus growth and Kordyukova and her colleagues have used bromelain
digestion to obtain information on the properties of M1. Identifying
peptide segments hidden within protein sequences, which are
termed cryptides, is described by Mukai and his colleagues
in their discussion of the effects of several peptides on
histamine release from mast cells. Kazuyasu Sakaguchi and
co-authors discuss a subfamily of the protein phosphatases,
the magnesium dependent phosphatases and use bioinformatic
methods to identify active site residues and to analyze substrate
specificity. The prion protein has an N-terminus that includes
four repeats of the peptide sequence Gly-Gln-Phe-His-Gly-Gly-Gly-Trp.
Shimohigashi and his colleagues analyze the amino acid composition
of different parts of the prion protein using the Radar Chart
method. An exciting new area of protein research involves
intrinsically disordered proteins. Vladimir Uversky, Keith
Dunker and their associates discuss the use of the TOP-IDP-Scale,
a new prediction tool for measuring the propensity for intrinsic
disorder. Willy Taylor and his colleagues Hollup and Jonassen
describe the use of protein fragments in protein modeling
and structure prediction. You-Ming Feng and his co-workers
report on studies of the equilibrium folding of the proinsulin
precursor protein. B. Moon Kim and his colleagues describe
the synthesis of a conformationally constrained γ-turn
mimic.
The final seven papers in this issue deal with protein expression,
purification, and analysis. Sue Douglas and coworkers describe
methods to express a group of small, highly disulphide-bonded,
cationic antimicrobial peptides. Mariana Castro, in collaboration
with colleagues Warner Fontes and C.F.M. Morris, discuss the
range of proteins present in the neutrophil proteome and review
other reports in this area. Snake venoms have been a rich
source of proteases, phospholipases, and toxins. Raghuvir
Arni and his colleagues discuss bothropstoxin-II, a myotoxic
Asp49 phospholipase A2 from
Bothrops jararacussu. Pedilanthus tithymaloids is
an ornamental lactiferous shrub with some medicinal properties.
Arvind M. Kayastha and his colleagues have purified a new
protease from the latex of this plant and named it pedilanthin.
A novel thermoacidophilic cellulase from Alicyclobacilllus
acidocaldarius is the topic of the report from Francesco La
Cara and his associates. A detailed analysis of the digestive
carbohydrases of the sixth instar of the meal moth may lead
to the development of advanced insecticides, as discussed
by M. P. Sales and his associates. PAT proteins are involved
in the regulation of triglyceride metabolism and Jose Soulages
and his associates have purified a PAT protein from Drosophila
melanogaster.
We plan to do one issue per year that will feature papers
from EAB members and have set a date for submission of manuscripts
for the 2009 special issue of October-November 2008.
Ben M. Dunn
Editor-in-Chief, Protein & Peptide Letters
Biochemistry and Molecular Biology
University of Florida, College of Medicine
Gainesville, FL 32610-0245
USA
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Article]
Targeting the Plasmepsin 4 Orthologs of Plasmodium
sp. with “Double Drug” Inhibitors
L. Janka, J. Clemente, N. Vaiana, A. Sparatore,
S. Romeo and B.M. Dunn
Plasmepsin 4 (PM4) is a digestive vacuole enzyme found
in all Plasmodium species examined to date. While
P. falciparum has three additional aspartic proteinases
in its digestive vacuole in addition to plasmepsin 4, other
Plasmodium species have only PM4 in their digestive
vacuole. Therefore, PM4 may be a good target for the development
of an antimalarial drug. This study presents data obtained
with PM4s from several Plasmodium species. Low nanomolar
Ki values have been observed
for all PM4s studied.
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Article]
Structural and Biochemical Investigation of Heptad Repeat
Derived Peptides of Human SARS Corona Virus (hSARS-CoV) Spike
Protein
S. Basak, X. Hao, A. Chen, M. Chrétien
and A. Basak
hSARS-CoV is the causative agent for SARS infection.
Its spike glycoprotein (S) is processed by host furin enzyme
to produce S1 and S2 fragments, the latter being crucial for
fusion with the host membrane. This takes place via formation
of a coiled coil 6-helix bundle involving N and C-terminal
heptad repeat domains (HR-N and HR-C) of S2. Several fluorescent
and non-fluorescent peptides from these domains were synthesized
to examine their interactions by circular dichroism, thermal
denaturation, native-page, mass spectrometry and fluorescence
spectroscopy studies. Data revealed that HR-C domains (1153-1189),
(1153-1172) and (1164-1184) all exhibit potent binding interactions
with HR-N892-931 domain.
These peptides may find useful therapeutic applications in
SARS intervention.
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Article]
Identification of a Thermo-Regulated Glutamine-Binding Protein
from Yersinia pestis
M. Cosman, J.B. Pesavento, A. Zemla, P.T.
Beernink, R. Balhorn and D. Barsky
Here we present modeling and NMR spectroscopic evidence
that the function of a Yersinia pestis pMT1 plasmid
protein, designated as orf38, is most likely a glutamine binding
protein. The modeling was homology-based at a very low level
of sequence identity (~ 16%) and involved structural comparison
of multiple templates, as well as template-substrate interaction
analyses. Transferred nuclear Overhauser and saturation transfer
difference experiments were used to characterize the binding
of sugars and amino acids to orf38. The identification and
characterization of an unknown protein function using the
strategy presented here has applicability to a variety of
research areas, including functional genomics and proteomics
efforts.
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Article]
Is Asparagine Deamidation in the Porcine Odorant-Binding Protein
Related to the Odor Molecules Binding?
G. Mamone and S. D’Auria
Odorant-binding proteins are biomolecules belonging to
the lipocalin family. Among all the odorant-binding proteins,
the porcine odorant-binding protein has been well characterized.
This protein is a monomer that is characterized by the presence
of the β-barrel
structure and of the disulphide bridge. The internal cavity
of the β-barrel
is the binding site. In this study we have investigated the
structural properties of the porcine odorant-binding protein
by mass spectrometry experiments. Our data allow us to hypothesize
that specific deamidation mechanisms of specific amino acid
residues can be responsible for the binding properties of
this class of proteins.
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Article]
Pt2L4 Protein, a Homologue to Hev b 5 from Rubber Tree, May
Not Be Responsible for the Cross-Reactions to Cassava Shown
by People Allergic to Latex
C.R.B. de Souza, D. Beezhold and
L.J.C.B. Carvalho
Pt2L4 is a protein from cassava homologue to Hevb5, a
principal allergen from latex. Here we aimed to elucidate
immunological relationships between these proteins. Our results
revealed that epitopes found in Hev b 5 are not entirely conserved
in Pt2L4 which is not recognized by IgE from patients allergic
to Hev b 5.
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Article]
Structural Refinement of Insecticidal Plant Proteinase Inhibitors
from Nicotiana alata
H.J. Schirra, M.A. Anderson and D.J.
Craik
Ornamental tobacco (Nicotiana alata) produces
a series of 6 kDa proteinase inhibitors belonging to the potato
type II inhibitor family. These proteins inhibit trypsin and
chymotrypsin, the main digestive enzymes of predatory insects,
thus leading to starvation, impaired larval development or
death. In this context, the three-dimensional structures of
these inhibitors are important for developing novel strategies
for pest control. The solution structures of C1 and T1, the
two main prototypes of the N. alata inhibitors, were
originally determined more than a decade ago (J. Mol.
Biol. 242, 231-243 (1994) and Biochemistry
34, 14304-14311 (1995)). Since then methods
for NMR structure calculations have evolved con-siderably.
Here we report the refinement of the structures of C1 and
T1 with state-of-the-art protocols for NMR structure calculations.
This refinement leads to an improved quality of the structures,
making them a more reliable basis for the development of novel
pesticides and modeling applications.
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Binding Mode of α-Conotoxins
to an Acetylcholine Binding Protein Determined by Saturation
Transfer Difference NMR
J.-C. Westermann, R.J. Clark and
D.J. Craik
The saturation transfer difference (STD) NMR technique
was employed to study the complex of the α-conotoxins
Vc1.1 and MII bound to the acetylcholine binding protein (AChBP)
from Lymnea stagnalis, a model system of the α7
subunit of the nicotinic acetylcholine receptor. MII was found
to be the more potent ligand for AChBP, consistent with data
from electrophysiology measurements for the nicotinic acetylcholine
receptor. Both peptides displayed strong interactions on aromatic
residues in the α-helical
part of their sequences, i.e., Tyr10 in Vc1.1 and His9 in
MII respectively. From the STD NMR spectra it was determined
that the peptides are buried in the nicotinic binding site
of ACBP as has been previously shown for the conotoxins PnIA[A10L,
D14K], ImI and TxIA[A10L] by X-ray crystallography. This study
demonstrates the value of STD NMR in the study of conotoxin
binding to receptor proteins.
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Predicting Membrane Protein Types by the LLDA Algorithm
T. Wang, J. Yang, H.-B. Shen and
K.-C. Chou
Membrane proteins are generally classified into the following
eight types: (1) type I transmembrane, (2) type II, (3) type
III, (4) type IV, (5) multipass transmembrane, (6) lipid-chain-anchored
membrane, (7) GPI-anchored membrane, and (8) peripheral membrane
(K.C. Chou and H.B. Shen: BBRC, 2007, 360: 339-345). Knowing
the type of an uncharacterized membrane protein often provides
useful clues for finding its biological function and interaction
process with other molecules in a biological system. With
the explosion of protein sequences generated in the Post-Genomic
Age, it is urgent to develop an automated method to deal with
such a challenge. Recently, the PsePSSM (Pseudo Position-Specific
Score Matrix) descriptor is proposed by Chou and Shen (Biochem.
Biophys. Res. Comm. 2007, 360, 339-345) to represent a protein
sample. The advantage of the PsePSSM descriptor is that it
can combine the evolution information and sequence-correlated
information. However, incorporating all these effects into
a descriptor may cause the “high dimension disaster”.
To overcome such a problem, the fusion approach was adopted
by Chou and Shen. Here, a completely different approach, the
so-called LLDA (Local Linear Discriminant Analysis) is introduced
to extract the key features from the high-dimensional PsePSSM
space. The dimension-reduced descriptor vector thus obtained
is a compact representation of the original high dimensional
vector. Our jackknife and independent dataset test results
indicate that it is very promising to use the LLDA approach
to cope with complicated problems in biological systems, such
as predicting the membrane protein type.
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Influenza A Virus M1 Protein Structure Probed by In Situ
Limited Proteolysis with Bromelain
L.V. Kordyukova, M.V. Serebryakova, V.Y.
Polyakov, T.V. Ovchinnikova, Yu. A. Smirnova, N.V. Fedorova
and L.A. Baratova
Influenza A virus matrix M1 protein is membrane associated
and plays a crucial role in virus assembly and budding. The
N-terminal two thirds of M1 protein was resolved by X-ray
crystallography. The overall 3D structure as well as arrangement
of the molecule in relation to the viral membrane remains
obscure. Now a proteolytic digestion of virions with bromelain
was used as an instrument for the in situ assessment
of the M1 protein structure. The lipid bilayer around the
subviral particles lacking glycoprotein spikes was partially
disrupted as was shown by transmission electron microscopy.
A phenomenon of M1 protein fragmentation inside the subviral
particles was revealed by SDS-PAGE analysis followed by in-gel
trypsin hydrolysis and MALDI-TOF mass spectrometry analysis
of the additional bands. Putative bromelain-digestion sites
appeared to be located at the surface of the M1 protein globule
and could be used as landmarks for 3D molecular modeling.
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Elucidation of Structural Requirements of Mastoparan for Mast
Cell Activation: Toward the Comprehensive Prediction of Cryptides
Acting on Mast Cells
H. Mukai, Y. Suzuki, Y. Kiso and
E. Munekata
Mastoparan, a toxic peptide from wasp venom, induces
various biological functions including histamine release from
rat peritoneal mast cells. Here we report that, for the activation
of mast cells by mastoparan, at least two positively charged
side chains are required on the hydrophilic side of the amphiphilic
structure of the peptide. The present results are expected
to be utilized for the bioinformatic and comprehensive identification
of endogenous mast cell-stimulating cryptides.
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Characterization of the Active Site and a Unique Uncompetitive
Inhibitor of the PPM1-Type Protein Phosphatase PPM1D
Y. Chuman, H. Yag, T. Fukuda, T. Nomura,
M. Matsukizono, Y. Shimohigashi and K. Sakaguchi
Protein phosphatase magnesium-dependent 1, delta (PPM1D)
is a member of the PPM1 (formerly PP2C) protein phosphatase
family, and is induced in response to DNA damage. The overexpression
of PPM1D is thought to exert oncogenic effects through the
inhibition of tumor suppressor proteins. PPM1D shows high
selectivity for the primary sequence in its substrates when
compared with other phosphatases, but the mechanisms underlying
substrate recognition by this enzyme is not clearly known.
In our present study we wished to identify the active center
and further elucidate the substrate preference of PPM1D, and
to this end performed sequence alignments among the human
PPM1 type phosphatases. The results of this analysis clearly
showed that the putative active site residues of PPM1D are
highly conserved among the PPM1 family members. Phosphatase
analyses using PPM1D mutants further identified the metal-chelating
residues and a phosphate binding residue. In kinetic analyses
using a series of phosphorylated p53 peptide analogs, the
introduction of acidic residues into the region flanking the
sites of dephosphorylation enhanced their affinity with PPM1D.
Homology modeling of PPM1D also revealed that PPM1D contains
two characteristic loops, a Pro-residue rich loop on the opposite
side of the active site and a basic-residue rich loop in the
vicinity of the active site in the catalytic domain. Interestingly,
nonhydrolyzable AP4-3E peptides derived from the acidic p53
peptide analogs very effectively blocked PPM1D activity in
an uncompetitive manner, suggesting that AP4-3E peptides may
be useful lead compounds in the development of novel inhibitors
of PPM1D.
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Radar Chart Deviation Analysis of Prion Protein Amino Acid
Composition Defines Characteristic Structural Abnormalities
of the N-Terminal Octapeptide Tandem Repeat
S. Yokotani, T. Nose, Y. Horiuchi, A. Matsushima
and Y. Shimohigashi
Analysis of the amino acid composition of prion protein
using a newly developed program for radar-chart deviation
analysis has identified an abnormality or irregularity of
the N-terminal flexible domain. Aromatic amino acids Trp and
His together with Gly are abnormally abounding in this N-terminal
domain, in which octapeptide GQPHGGGW is connected four times
in tandem. This tetrarepeat structure has been suggested to
be essential for the prion protein not only to play an intrinsic
functional role in the physiological condition, but also to
bring on structural abnormalities in prion disease.
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TOP-IDP-Scale: A New Amino Acid Scale Measuring Propensity
for Intrinsic Disorder
A. Campen, R.M. Williams, C.J. Brown, J.
Meng, V.N. Uversky and A.K. Dunker
Intrinsically disordered proteins carry out various biological
functions while lacking ordered secondary and/or tertiary
structure. In order to find general intrinsic properties of
amino acid residues that are responsible for the absence of
ordered structure in intrinsically disordered proteins we
surveyed 517 amino acid scales. Each of these scales was taken
as an independent attribute for the subsequent analysis. For
a given attribute value X, which is averaged over
a consecutive string of amino acids, and for a given data
set having both ordered and disordered segments, the conditional
probabilities P(s0
| x) and p(sd
| x) for order and disorder, respectively,
can be determined for all possible values of X. Plots of the
conditional probabilities P(s0
| x) and p(sd
| x) versus X give a pair of curves.
The area between these two curves divided by the total area
of the graph gives the area ratio value (ARV), which is proportional
to the degree of separation of the two probability curves
and, therefore, provides a measure of the given attribute’s
power to discriminate between order and disorder. As ARV falls
between zero and one, larger ARV corresponds to the better
discrimination between order and disorder. Starting from the
scale with the highest ARV, we applied a simulated annealing
procedure to search for alternative scale values and have
managed to increase the ARV by more than 10%. The ranking
of the amino acids in this new TOP-IDP scale is as follows
(from order promoting to disorder promoting): W, F, Y, I,
M, L, V, N, C, T, A, G, R, D, H, Q, K, S, E, P. A web-based
server has been created to apply the TOP-IDP scale to predict
intrinsically disordered proteins (http://www.disprot.org/dev/disindex.php).
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Structural Fragments in Protein Model Refinement
S.M. Hollup, W.R. Taylor and I.
Jonassen
We survey a method that uses patterns of residue packing
in known protein structures to refine structural models. The
method can be used to refine models that include only one
coordinate point per residue (Cα)
and is not dependent on homology. We demonstrate that the
method improves both decoy and CASP7 target models.
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Equilibrium Folding of Porcine Insulin Precursor in the Presence
of Redox Buffer: Implications for the Common Intermediates
Shared by Its Unfolding/Refolding Processes
J. Zhao, Q.-L. Huang, Y.-H. Tang, Z.-Y.
Guo, Z.-S. Qiao, G.-J. Xu and Y.-M. Feng
We use the procedure established for ‘disulfide
stability analysis in redox system’ to investigate the
unfolding process of porcine insulin precursor (PIP). Six
major unfolding intermediates have been captured, in which
four contain two disulfides, two contain one disulfide. Based
on the characterization and analysis of the intermediates
an unfolding pathway has been proposed, by which the native
PIP unfolded through in turn 2SS and 1SS intermediates into
fully reduced form. Besides, the comparison of the intermediates
captured in PIP unfolding process with those intermediates
captured in its refolding process revealed that some intermediates
captured during both unfolding/refolding processes of PIP
have identical disulfide pairing pattern, from which we suggest
that the unfolding/refolding processes of PIP share some common
intermediates but flow in the opposite direction.
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Synthesis and Structural Analysis of 6-Aminobicyclo[2.2.1]heptane-2-carboxylic
Acid as a Conformationally Constrained γ-Turn
Mimic
J.-S. Park, K.R. Kim, H.Y. Nam, C.-E. Yeom,
C. Chough, S.H. Kwon, S. Ro, D.-K. Shin and B.M.
Kim
An efficient asymmetric synthesis of 6-aminobicyclo[2.2.1]heptane-2-carboxylic
acid as a novel γ-turn
mimic has been achieved. Structural analysis of the γ-amino
acid derivative was carried out using 1H
NMR spectroscopy and intramolecular hydrogen bonding between
side chain amides confirmed the turn structure, which had
been predicted by Ab initio computational study.
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Strategies for Recombinant Expression of Small, Highly Disulphide-Bonded,
Cationic Antimicrobial Peptides
A.L. Greenshields, L.C. Knickle, R. Syvitski
and S.E. Douglas
Expression of two recombinant hepcidin homologues from
Atlantic salmon, Salmo salar, characterization of
their antimicrobial activity, and partial structural determination
of the peptides is described. Expression was attempted in
baculovirus and bacterial expression systems and the various
purification and refolding methods used to determine the optimal
strategy for production of active, correctly refolded hepcidin
are reviewed.
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Neutrophil Proteome: Lessons from Different Standpoints
C.F.M. Morris, M.S. Castro and
W. Fontes
The present review brings a timeline of some of the major
steps given throughout the years towards the development of
our knowledge regarding the biology of the neutrophil. The
contribution of early articles and their elementary biochemical
approach is highlighted. The importance of the development
of proteomic techniques is paralleled to the shift in neutrophil
research towards high throughput molecular methods. As a last
change of standpoint, the study of the neutrophil is presented
integrated with other life- sciences technologies such as
lipidomics, genomics and systems biology. The paper also brings
a perspective/tendency overview at the same time that it discusses
some of the difficulties encountered in the research of the
neutrophil.
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Biochemical and Structural Investigations of Bothropstoxin-II,
a Myotoxic Asp49 Phospholipase A2
from Bothrops jararacussu Venom
M.T. Murakami, M.R. Lourenzoni, E.Z. Arruda,
M.A. Tomaz, M.M. Viçoti, J.R.B. Abrego, P.A. Melo and
R.K. Arni
Bothropstoxin-II a calcium-dependent enzyme from Bothrops
jararacussu venom causes tissue damage and several haemostatic
disorders including platelet aggregation. In order to elucidate
the structural determinants of its multiple pharmacological
activities, we have studied the effects of suramin on Bothropstoxin-II
and present details concerning the mode of binding.
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Purification and Characterization of a Novel Protease
from the Latex of Pedilanthus tithymaloides
R. Bhowmick, N.K.P. Kumari, M.V. Jagannadham
and A.M. Kayastha
A novel protease was purified to homogeneity from the
latex of Pedilanthus tithymaloids by a simple purification
procedure involving ammonium sulfate precipitation and cation-exchange
chromatography. The molecular weight of the protease was estimated
to be approximately 63.1 kDa and the extinction coefficient
(ε1%280nm)
was 28.4. The enzyme hydrolyzes denatured natural substrates
like casein, azoalbumin and azocasein with a high specific
activity but little activity towards synthetic substrates.
The pH and temperature optima were pH 8.0-9.5 and 65-70 °C,
respectively. The proteolytic activity of the enzyme was inhibited
by different protease-specific inhibitors (e.g., thiol, serine,
metallo, etc.) up to a certain extent but not completely by
any class of inhibitors. The enzyme was relatively stable
towards pH change, temperature, denaturants and organic solvents.
The enzyme consists of five disulfide bridges compared to
three observed in most plant cysteine proteases. Overall,
the striking features of this protease are its high molecular
weight, high cysteine content and only partial inhibition
of activity by different classes of protease inhibitors contrary
to known proteases from other plant sources. The enzyme is
named as pedilanthin as per the protease nomenclature.
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A Novel Thermoacidophilic Cellulase from Alicyclobacillus
acidocaldarius
A. Morana, A. Esposito, L. Maurelli, G.
Ruggiero, E. Ionata, M. Rossi and F. La Cara
A novel cellulase was isolated from the thermoacidophilic
bacterium Alicyclobacillus acidocaldarius ATCC27009
grown in medium containing carboxymethylcellulose. The enzyme
is a glycosylated monomer of 56.2 kDa, relatively thermostable,
with optimal pH and temperature of 4.0 and 65 °C,
respectively. Enzymatic assays on several polysaccharides
demonstrated that CelG was specific for carboxymethylcellulose.
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Major Digestive Carbohydrase During Larval Development of
Meal Moth, Plodia interpunctella (Lepidoptera: Pyralidae)
M.P. de Sales, A. Alcazar, L.M. Lima, T.M.L.
Amorim, J.C.M. Pitanga, R.A. Pereira, L.L.P. Macedo, F.P.
Macedo, A.S. Oliveira and A.F. Uchôa
The digestive system of P. interpunctella was
characterized during its larval development to determination
of carbohydrases using disaccharides (sucrose and maltose)
and polysaccharides (starch and inulin) as substrate. At 6th
instar larval, Invertase>α-amylase>
maltase activities peaks were observed. Invertase was fractionated
with acetone and isolated. The Invertase was 485.5 fold purified
by Sephacryl S-200 and DEAE-Sephadex. Its kinetic parameters
were Km of 6.6 mM, Vmax
of 0.48, pH optimum of 5.5 and temperature optimum of 30 °C.
This enzyme was activated by CaCl2
and inhibited by EDTA. When analyzed by SDS-PAGE it showed
one band of Mr 34 kDa. The
understanding of the digestive system of P. interpunctella
could be a key step in the design of bioinsecticides.
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Purification and Characterization of Recombinant Lipid Storage
Protein-2 from Drosophila melanogaster
E.L. Arrese, L. Rivera, M. Hamada and
J.L. Soulages
Lipid storage protein 2 (Lsd 2) is a conserved insect
protein that belongs to the small PAT family of proteins.
PAT proteins are found associated to the lipid droplets of
adipocytes and play significant roles in the regulation of
triacylglycerides metabolism. Here we describe the expression
and purification of Lsd2, its reconstitution in lipoprotein
particles, the location of putative lipid binding sites and
its secondary structure. This study provides the starting
point for future studies on the mechanism of function of Lsd2.
The similarities and differences between Lsd1 and Lsd2, the
only PAT proteins found in insects, are discussed.
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