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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 13, Number 9, 2006
Contents
Regular Papers
Multibranch and Pseudopeptide Approach for Design
of Novel Inhibitors of Subtilisin Kexin Isozyme-1 Pp.
863-876
S. Basak, D. Mohottalage and A. Basak
[Abstract] [Purchase
Article]
Interaction Partners of the PDZ Domain of Erbin
Pp. 877-881
A. Ress and K. Moelling
[Abstract] [Purchase
Article]
Snapshots of Protein Folding Problem: Implications
of Folding and Misfolding Studies Pp. 883-888
V.K. Dubey, M. Pande and M.V. Jagannadham
[Abstract] [Purchase
Article]
Stereo-Structural Prediction and IR-Characteristic
Band Assignment of Amorphous Protonated Forms of Homodipeptides
L-Phe-L-Phe, L-Trp-L-Trp,
L-Tyr-L-Tyr and the
Neutral -L-Trp-L Trp.
Ab Initio Approximation and Solid-State Linear-Polarized IR
Spectroscopy Pp. 889-896
B.B. Ivanova and M.G. Arnaudov
[Abstract] [Purchase
Article]
A Novel Antiproliferative and Antifungal Lectin
from Amaranthus viridis Linn Seeds Pp. 897-905
N. Kaur, V. Dhuna, S.S. Kamboj, J.N. Agrewala and J. Singh
[Abstract] [Purchase
Article]
Isolation of Influenza Virus A Hemagglutinin
C-Terminal Domain by Hemagglutinin Proteolysis in Octylglucoside
Micelles Pp. 907-913
V.A. Radyukhin, M.V. Serebryakova, A.L. Ksenofontov, E.V.
Lukashina and L.A. Baratova
[Abstract] [Purchase
Article]
Expression of Human Tyrosine Kinase, Lck, in
Yeast Saccharomyces cerevisiae: Growth Suppression
and Strategy for Inhibitor Screening Pp. 915-920
M. Koyama, S. Saito, R. Nakagawa, I. Katsuyama, M. Hatanaka,
T. Yamamoto, T. Arakawa and M. Tokunaga
[Abstract] [Purchase
Article]
Aggregation Suppression of Proteins by Arginine
During Thermal Unfolding Pp. 921-927
T. Arakawa, Y. Kita, D. Ejima, K. Tsumoto and H. Fukada
[Abstract] [Purchase
Article]
Crystallization Reports
Statistical Analysis of 15 Dimensions in the Crystallization
Space for Protein-DNA Complexes Pp. 929-939
F. Saïda
[Abstract] [Purchase
Article]
Preparation, Crystallization and Preliminary
X-Ray Analysis of the Fab Fragment of Monoclonal Antibody
MN423, Revealing the Structural Aspects of Alzheimer’s
Paired Helical Filaments Pp. 941-944
N. Csóková, R. Skrabana, L. Urbániková,
B. Kovácech, A. Popov, J. Sevcík and M. Novák
[Abstract] [Purchase
Article]
Expression, Purification, and Preliminary X-Ray
Crystallographic Analysis of the Complex of Gαi3-RGS5
from Human with GDP/Mg2+/AlF4-
Pp. 945-949
K.-H. Rhee, K.-H. Nam, W.-H. Lee, Y.-G. Ko, E.E. Kim and
K.Y. Hwang
[Abstract] [Purchase
Article]
Crystallization and Preliminary X-Ray Analysis
of the Catalytic Domain of Chitinase D from Bacillus circulans
WL-12 Pp. 951-954
Y. Kezuka, K. Bando, H. Kobayashi, Y. Yonou, T. Sato,
T. Watanabe and T. Nonaka
[Abstract] [Purchase
Article]
Protein Expression, Crystallization and Preliminary
X-Ray Crystallographic Studies on HSCARG from Homo Sapiens
Pp. 955-957
X. Dai, X. Gu, M. Luo and X. Zheng
[Abstract] [Purchase
Article]
Abstracts
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Multibranch and Pseudopeptide Approach for Design
of Novel Inhibitors of Subtilisin Kexin Isozyme-1
S. Basak, D. Mohottalage and A. Basak
Here we developed small molecule inhibitors of SKI-1/S1P enzyme
of the Proprotein Convertase family following two approaches.
One involves the assembly of multi-branch peptides while the
other utilizes the insertion of alkyloxy pseudo peptide bond
at P1-P1’ cleavage position. In first approach, 2 and
4 branch peptides were designed based on the human (h) SKI-1128-137
sequence, located N-terminal to its secondary activation site
(K137 L).
The 4-branch peptide exhibited the highest SKI-1 inhibitory
property (IC50 = 0.9 μM)
with ~8.6 and 1.3-fold more potency than the corresponding
single and 2 branch peptides, respectively. In the second
strategy, an oxymethylene containing unnatural amino acid
such as aminooxy acetic acid (Aoaa) or 8-amino-3,
6 dioxa-octanoic acid (Adoa) was introduced
substituting P1, P1’ or both residues of hSKI 1183-190
and hSKI-1178-190 segments. These domains
contain the same primary hSKI-1 activation site L186↓
R. Among those tested, P7-Tyr mutant [178GRYSSRRL(Adoa)AIP190]
exhibited higher SKI-1 inhibitory activity (Ki in low mM).
Circular dichroism (CD) spectra of SKI-1 inhibitors showed
interactions of varying degrees between the enzyme and the
inhibitor consistent with the observed inhibition profile.
A 3D-homology model structure of SKI-1 catalytic domain indicated
a broad catalytic pocket.
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Interaction Partners of the PDZ Domain of Erbin
A. Ress and K. Moelling
In order to identify proteins that bind to the PDZ domain
of Erbin, we tested the C-termini of several proteins in a
yeast two-hybrid assay. ErbB2, APC, β-catenin,
c-Rel and HTLV-1 Tax were identified as ligands of the PDZ
domain of Erbin. The interactions were verified by co-immunoprecipitation
experiments. These findings demonstrate the promiscuity of
the PDZ domain of Erbin.
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Snapshots of Protein Folding Problem: Implications
of Folding and Misfolding Studies
V.K. Dubey, M. Pande and M.V. Jagannadham
Deciphering the code that determines the three-dimensional
structure of proteins and the ability to predict the final
folded form of a protein is still elusive to molecular biophysists.
In the case of several proteins a similar tertiary structure
is not accompanied by any significant sequence similarity.
The question now remains whether a code beyond the genetic
code that describes the arrangement of the amino acid within
a three dimensional protein structure. The available data
undoubtedly demonstrates that the redundancy of this code
must be tremendous. Several techniques such as nuclear magnetic
resonance spectroscopy and laser detection techniques, coupled
with fast initiation of the folding reaction, can now probe
the folding events in milliseconds or even faster and provide
highly relevant information. The thermodynamic analysis of
the folding process and of kinetic intermediates opens whole
new avenue of understanding. Breaking the protein folding
code would enable scientists to look at a gene whose function
is unknown and predict the three-dimensional structure of
the protein it encodes. This would give them a very good idea
of what the gene does. In this review we hope to bring together
the information available about protein folding with particular
emphasis on folding intermediate(s). Additionally, the practical
consequences of the solution of the protein folding problem
in medicine and biotechnology are also discussed.
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Stereo-Structural Prediction and IR-Characteristic
Band Assignment of Amorphous Protonated Forms of Homodipeptides
L-Phe-L-Phe, L-Trp-L-Trp,
L-Tyr-L-Tyr and the
Neutral -L-Trp-L Trp.
Ab Initio Approximation and Solid-State Linear-Polarized IR
Spectroscopy
B.B. Ivanova and M.G. Arnaudov
Stereo-structures of protonated L-phenylalanine
(L-Phe), L-tyrosine
(L-Tyr) and L-tryptophan (L-Trp)
containing homodipeptides (L-Tyr-L-Tyr,
L-Phe-L-Phe, L-Trp-L-Trp)
are carried out by ab initio calculations. The obtained data
in gas phase are compared with experimental ones, received
by linear-dichroic infrared (IR-LD) spectroscopy of solids,
oriented as suspension in nematic mesophase. An observation
of a good correlation between theoretical and spectroscopic
geometry parameters established and illustrated the possibilities
of this complex study approach for the prediction of the stereo-structures
in compounds in solid state. The protonation leads to little
variance in the bond lengths and angles, expecting the COO-
fragment, where a distortion of equalized COO-
bond lengths, stabilizing a C=O double bond and C-O(H) one
is established. Significant deviations of the dihedral angles
as a result of the protonation are obtained in the skeletal
aliphatic and amide- fragments. In L-Tyr-L-Tyr
and L-Phe-L-Phe, a
deviation of O=C-N-H torsion angle about 10-140
is predicted. The calculations show a trans-amide
configuration in L-Trp-L-Trp
and a cis-one after its protonation.
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A Novel Antiproliferative and Antifungal Lectin
from Amaranthus viridis Linn Seeds
N. Kaur, V. Dhuna, S.S. Kamboj, J.N. Agrewala and J. Singh
A lectin from the seeds of Amaranthus viridis
Linn has been purified by affinity chromatography on asialofetuin-linked
amino activated silica. Amaranthus viridis lectin
(AVL) has a native molecular mass of 67 kDa. It is a homodimer
composed of two 36.6 kDa subunits. The lectin gave a single
band in non-denaturing PAGE at pH 4.5 and pH 8.3 and a single
peak on HPLC size exclusion and cation exchange columns. The
purified lectin was specific for both T-antigen and N-acetyl-D-lactosamine,
markers for various carcinomas, in addition to N-acetyl-D-galactosamine,
asialofetuin and fetuin. This lectin reacted strongly with
red blood cells (RBCs) from human ABO blood groups and rat.
It also reacted with rabbit, sheep, goat and guinea pig RBCs.
The lectin is a glycoprotein having no metal ion requirement
for its activity. Denaturing agents such as urea, thiourea
and guanidine-HCl had no effect on its activity when treated
for 15 minutes. AVL showed significant antiproliferative activity
towards HB98 and P388D1 murine cancer cell lines. It also
exerted antifungal activity against phytopathogenic fungi
Botrytis cincerea and Fusarium oxysporum
but not against Rhizoctonia solani, Trichoderma reesei,
Alternaria solani and Fusarium graminearum.
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Isolation of Influenza Virus A Hemagglutinin
C-Terminal Domain by Hemagglutinin Proteolysis in Octylglucoside
Micelles
V.A. Radyukhin, M.V. Serebryakova, A.L. Ksenofontov, E.V.
Lukashina and L.A. Baratova
A method of isolation of hydrophobic membrane-bound C-terminal
domain of influenza virus A hemagglutinin (HA) is suggested.
The method is based on the insertion of HA into octylglucoside
micelles followed by pepsin or thermolysin hydrolysis. Subsequent
treatment of proteolytic digests with chloroform-hexafluoroisopropanol
mixture resulted in the extraction of a few hydrophobic peptides
into organic phase. Mass-spectrometry (MALDI-TOF) analysis
revealed that the peptides with ion masses corresponding to
the anchoring C-terminal domain with or without modifications
predominated in the organic solution. The data obtained confirmed
our speculation on the possibility of the suggested isolation
scheme following from the strong interactions of anchoring
domains in compact trimeric structure of HA spikes combined
with micelle protection effect. Several appropriate peptides
presence in the organic phase apparently arises from the presence
of a few accessible proteolytic sites in HA transmembrane
region.
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Expression of Human Tyrosine Kinase, Lck, in
Yeast Saccharomyces cerevisiae: Growth Suppression
and Strategy for Inhibitor Screening
M. Koyama, S. Saito, R. Nakagawa, I. Katsuyama, M. Hatanaka,
T. Yamamoto, T. Arakawa and M. Tokunaga
We report the successful expression and detection of
a phosphorylated form of human T cell tyrosine kinase, Lck,
in Saccharomyes cerevisiae, which leads to growth
suppression of the yeast cells. Expression of an inactive
Lck mutant resulted in no phosphorylation and no growth suppression,
indicating that cell growth inhibition by Lck is due to the
activity of the kinase, consistent with the observed tyrosine-phosphorylation
of the Lck and yeast host cell proteins. The addition of a
known inhibitor of Lck to the cell culture resulted in recovery
of cell growth expressing the active Lck, suggesting that
the growth inhibition by lck gene expression can
be used to screen inhibitors for the gene product. We have
extended such approach to Tob, another potential therapeutic
target.
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Aggregation Suppression of Proteins by Arginine
During Thermal Unfolding
T. Arakawa, Y. Kita, D. Ejima, K. Tsumoto and H. Fukada
Arginine has been used to suppress aggregation of proteins
during refolding and purification. We have further studied
in this paper the aggregation-suppressive effects of arginine
on two commercially important proteins, i.e., interleukine-6
(IL-6) and a monoclonal antibody (mAb). These proteins show
extensive aggregation in aqueous buffers when subjected to
thermal unfolding. Arginine suppresses aggregation concentration-dependently
during thermal unfolding. However, this effect was not specific
to arginine, as guanidine hydrochloride (GdnHCl) at identical
concentrations also was effective. While equally effective
in aggregation suppression during thermal unfolding, arginine
and GdnHCl differed in their effects on the structure of the
native proteins. Arginine showed no apparent adverse effects
on the native protein, while GdnHCl induced conformational
changes at room temperature, i.e., below the melting temperature.
These additives affected the melting temperature of IL-6 as
well; arginine increased it concentration-dependently, while
GdnHCl increased it at low concentration but decreased at
higher concentration. These results clearly demonstrate that
arginine suppresses aggregation via different mechanism from
that conferred by GdnHCl.
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Statistical Analysis of 15 Dimensions in the Crystallization
Space for Protein-DNA Complexes
F. Saïda
Solving the three dimensional structure of a protein-DNA
complex is a prerequisite to understand, at the atomic level,
the interactions between DNA-binding proteins and their target
DNA sequences. Arranging these complexes into an ordered and
repetitive network (a crystal, suitable for X-Ray analysis)
is a time-limiting empirical step. Although it has been suggested
that the crystallization space for protein-DNA complexes is
probably smaller than that of non-complexed proteins, a study
presenting a detailed and updated analysis of this space is
still missing. Here, we analyze the successful crystallization
conditions of several hundred protein-DNA complexes and present
a bias-free statistical analysis of 15 crystallization parameters
that include concentration, temperature, pH, precipitants,
salts, divalent cations and polyamines. Our analysis shows
that some crystallization parameters are interestingly restricted
into narrow intervals. These restrictions could be very helpful
in the design of sparse-matrix crystallization screens that
target exclusively protein-DNA complexes.
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Preparation, Crystallization and Preliminary
X-Ray Analysis of the Fab Fragment of Monoclonal Antibody
MN423, Revealing the Structural Aspects of Alzheimer’s
Paired Helical Filaments
N. Csóková, R. Skrabana, L. Urbániková,
B. Kovácech, A. Popov, J. Sevcík and M. Novák
Monoclonal antibody (mAb) MN423 recognizes Alzheimer’s
disease specific conformation of tau protein assembled into
paired helical filaments (PHF). Since the three-dimensional
structure of PHF is currently unavailable, the structure of
MN423 binding site could provide important information about
PHF conformation with the consequences for the Alzheimer’s
disease prevention and cure. Fab fragment of MN423 was prepared
and purified. We have identified two different conditions
for crystallization of the Fab fragment that yielded two crystal
forms. They diffracted to 3.0 and 1.6 Å resolution with
four and one molecule in the asymmetric unit, respectively.
Both crystal forms belonged to the space group P21
with unit cell parameters a = 76.4 Å, b = 138.4 Å,
c = 92.4 Å, β
= 101.90, and a = 71.5 Å, b = 36.8 Å,
c = 85.5 Å, β
= 113.90.
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Expression, Purification, and Preliminary X-Ray
Crystallographic Analysis of the Complex of Gαi3-RGS5
from Human with GDP/Mg2+/AlF4-
K.-H. Rhee, K.-H. Nam, W.-H. Lee, Y.-G. Ko, E.E. Kim and
K.Y. Hwang
Regulator of G-protein signaling 5 (RGS5), an inhibitor
of Gq and Gi activation, is a member of
the small RGS protein subfamily. However, despite significant
process in the investigation of RGS5, no structure is yet
available. In order to elucidate the mechanism of the RGS5
in G protein signaling pathway, we have overexpressed the
RGS5 and Gαi3
from human in Escherichia coli and crystallized the
complex of RGS5 and Gαi3
proteins with GDP/Mg2+/AlF4- at 3.0
Å resolution using a synchrotron radiation source. The
complex crystals belong to the tetragonal space group P41212
or P43212, with unit cell parameters
a=b=95.9 Å, and c=138.8 Å. Assuming one complex
protein in the crystallographic asymmetric unit, the calculated
Matthews parameter (VM) is 2.57 Å3/Da
and solvent content is 52.2 %.
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Crystallization and Preliminary X-Ray Analysis
of the Catalytic Domain of Chitinase D from Bacillus circulans
WL-12
Y. Kezuka, K. Bando, H. Kobayashi, Y. Yonou, T. Sato,
T. Watanabe and T. Nonaka
We report here on crystallization and preliminary X-ray
analysis of the catalytic domain of chitinase D from Bacillus
circulans WL-12. The native crystals of this domain were
found to belong to the orthorhombic space group P212121.
To elucidate the structure of the catalytic domain by the
multiple isomorphous replacement method, 30 kinds of derivatized
crystals were prepared by soaking the native crystals into
a mother liquor containing salts of heavy metal atoms. Difference
Patterson maps calculated for four derivatives showed strong
peaks in the Harker sections.
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Protein Expression, Crystallization and Preliminary
X-Ray Crystallographic Studies on HSCARG from Homo Sapiens
X. Dai, X. Gu, M. Luo and X. Zheng
Human HSCARG has been annotated as a possible cancer related
protein. Amino acid homology, although at a low percentage,
suggested that HSCARG contains NmrA domain and might be a
member of short chain dehydrogenase reductase superfamily.
In order to investigate its structure and function, HSCARG
gene has been successfully expressed and purified in E.
coli. HSCARG was crystallized and diffracted to a resolution
of 2.4 Å on Mar225 CCD Detector at SER-CAT 22BM synchrotron
source. The crystals belong to F23 space group, with unit
cell parameters a=b=c=223.30Å, α=β=γ=900.
There are two molecules per asymmetry unit.
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