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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 13, Number 6, 2006
Contents
Regular Papers
Investigation of Kinetic Parameters In Vitro
of Serine Proteinases Included in the Blood Coagulation Cascade
Pp. 535-537
D. Danalev, L. Yotova and L. Vezenkov
[Abstract]
Tyrosine Residues of the Extrinsic 23 kDa Protein
Are Important for Its Interaction with Spinach PSII Membranes
Pp. 539-544
J. Gao, F. Zhang, J. Weng, K. Ruan and C. Xu
[Abstract]
Effect of Organic Solvents on the Molten Globule
State of Procerain: β-Sheet to α-Helix Switchover
in Presence of Trifluoroethanol Pp. 545-547
V.K. Dubey, A. Shah, M.V. Jagannadham and A.M. Kayastha
[Abstract]
A Linear Function for the Approximation of Accessible
Surface Area of Proteins Pp. 549-553
R.G. Kim and C.-Y. Choi
[Abstract]
Non-Additive Counteraction of KCl-Perturbation
of Lactate Dehydrogenase by Trimethylamine N-Oxide
Pp. 555-557
M.K. Desmond and J.F. Siebenaller
[Abstract]
Expression of Soluble and Functional Snake Venom
Fibrinolytic Enzyme Fibrolase Via the Co-Expression
of DsbC in Escherichia coli Pp. 559-563
S.-T. Zhang, J. Shi, J. Zhao, Y.-F. Qi and A.-G. Guo
[Abstract]
Kinetics of Inactivation of Phytase (phy A) During
Modification of Histidine Residue by IAA and DEP
Pp. 565-570
X.-Y. Wang, M.-L. Sun, D.-M. Zhao and M. Wang
[Abstract]
cDNA Cloning, Purification and Properties of Paralithodes
camtschatica Metalloprotease Pp. 571-575
S.A. Semenova, G.N. Rudenskaya, D.V. Rebrikov and V.A.
Isaev
[Abstract]
Juxtaposed Half-Cystines as Disulphide Bridged Partners
in Protein Tertiary Structure Pp. 577-579
K. Guruprasad, V.J. Kartik, T. Lavanya and L. Guruprasad
[Abstract]
Spectroscopic Studies on Erythrina lysistemon
Seed Lectin Effect of Denaturing Agents on Lectin: Quaternary
Structure Pp. 581-586
E.H.E. Konozy, M.B. Dafalla and C.-D. Hsiao
[Abstract]
Data Mining of VDJ Genes Reveals Interesting Clues
Pp. 587-593
R.R. Joshi and V.K. Gupta
[Abstract]
The Prediction of Hydrophobicity Gradients within
Transmembrane Protein α-Helices
Using a Novel Graphical Technique Pp. 595-600
F. Harris, S. Dennison and D.A. Phoenix
[Abstract]
Determination of Mutation Trend in Hemagglutinins
by Means of Translation Probability Between RNA Codons and
Mutated Amino Acids Pp. 601-609
G. Wu and S. Yan
[Abstract]
Efficient Expression of Haloarchaeal Nucleoside Diphosphate
Kinase Via Strong Porin Promoter in Moderately Halophilic
Bacteria Pp. 611-615
C. Nagayoshi, H. Tokunaga, A. Hayashi, H. Harazono, K.
Hamasaki, A. Ando and M. Tokunaga
[Abstract]
Characterization of Bovine Tau-Preparations by
Two-Dimensional Gel Electrophoresis Pp. 617-625
D. Volke and R. Hoffmann
[Abstract]
Crystallization Reports
Crystallization and Preliminary X-Ray Analysis of
Sau3AI C-Terminal 233-419 Fragment Pp. 627-628
F. Yu, J. Song, C. Xu, Y. Ding, X. Hu, J. He and Z. Zhang
[Abstract]
Crystallization and Preliminary X-Ray Diffraction
Analysis of Three Mastoparans Pp. 629-631
F. Wang, X. Liu, H. Li, X. Lang, H. Peng, S. Liu and T.
Jiang
[Abstract]
Zebrafish Caspase-3: Molecular Cloning, Characterization,
Crystallization and Phylogenetic Analysis Pp. 633-640
C. Chakraborty, S.S. Nandi, S. Sinha and V.K. Gera
[Abstract]
Expression, Purification, Crystallization and
Preliminary Crystallographic Analysis of the Human Intracellular
Chloride Channel Protein CLIC4 Pp. 641-643
D.-F. Li, Y.-F. Li, Q.-H. Huang, Y. Zhang and D.-C. Wang
[Abstract]
Abstracts
[Back to top]
Investigation of Kinetic Parameters In Vitro
of Serine Proteinases Included in the Blood Coagulation Cascade
D. Danalev, L. Yotova and L. Vezenkov
During the last years the cases and death due to hemostatic
violations exceed that of tumors. Enormous efforts have devoted
to the prevention and treatment of some diseases such as arterial
thrombosis. Antistasin, a 15 kDa anticoagulant protein isolated
from salivary glands of the Mexican leech Haementeria
officinalis, has been shown to be a potent inhibitor
of Factor Xa in the blood coagulation cascade. Some short
analogues which are hybrid structure between isoform 2 and
3 of antistasin and tripeptides inhibitors of serine proteinases
were synthesized and reported in our previous work. Inhibitor
constants, mechanism, and type of inhibition of some short
analogues of antistasin are investigated. These analogs which
show high anticoagulant activity in vitro in pure
platelet human plasma.
[Back to top]
Tyrosine Residues of the Extrinsic 23 kDa Protein
Are Important for Its Interaction with Spinach PSII Membranes
J. Gao, F. Zhang, J. Weng, K. Ruan and C. Xu
The 1, 4, and 8 tyrosine (Tyr) residues on the PSII extrinsic
23 kDa protein were modified with 5, 10 or 40 mM N-acetylimidazole
(NAI) respectively. The amount of rebound NAI-modified extrinsic
23 kDa protein was 98%, 80%, and 5% of that in the unmodified
protein, respectively. These results indicate that the Tyr
residues are absolutely essential to reconstitution ability.
Further, the fluorescence and circular dichroism spectra among
native and NAI-modified extrinsic 23 kDa proteins were similar,
suggesting that the modification by NAI did not markedly influence
the basic secondary structure of the native conformation.
Thus, we have concluded that the tyrosine residues in the
extrinsic 23 kDa protein are important for interaction with
PSII membranes. In addition, we found that the structure of
the extrinsic 23 kDa protein is stable in suspension (pH 4-9
or Tm 25-55°C).
[Back to top]
Effect of Organic Solvents on the Molten Globule
State of Procerain: β-Sheet to αHelix Switchover
in Presence of Trifluoroethanol
V.K. Dubey, A. Shah, M.V. Jagannadham and A.M. Kayastha
The effect of methanol and trifluoroethanol (TFE) on the structure
and folding of molten globule state of procerain, a cysteine
protease from Calotropis procera, was studied by
circular dichroism spectroscopy. The magnitude of ellipticity
at 215 nm, as a measure of β
-sheet content, is dependent on the concentration of the TFE.
Interestingly, a switch over from the β
-sheet structure of the molten globule state to α
-helix was observed at 60% TFE and the ellipticity at 222
nm increased as a function of TFE concentration beyond this
critical TFE concentration. Temperature induced unfolding
of the molten globule state of procerain in 10% methanol showed
stabilization of α
-rich domain with concomitant destabilization of β
-rich domain. Using higher concentration of methanol (20-40
%) had no stabilizing effect on the α-rich
domain however, the β-rich
domain was destabilized, indicating that the stability of
the domains were not interdependent and that a low concentration
of methanol induced stabilization in α-rich
domain.
[Back to top]
A Linear Function for the Approximation of Accessible
Surface Area of Proteins
R.G. Kim and C.-Y. Choi
Since the solvent accessible surface area of proteins has
been utilized as an important factor in the prediction of
their thermodynamic properties, a simple and analytical method
for its determination would contribute to protein structure
prediction and analysis methods. We report the development
of a simple and analytical method for the estimation of the
accessible surface area of protein, consisting of linear functions
of distances between unified atoms. While our formulation
was much simpler than previously developed methods, it showed
comparable performance, with the mean relative error on total
solvent accessible surface area of 0.49 and 2.16% for 89 denatured
and native protein structures, respectively. Moreover, the
partial derivatives of accessible surface area to the position
of unified atoms could also be derived in a simpler form.
[Back to top]
Non-Additive Counteraction of KCl-Perturbation
of Lactate Dehydrogenase by Trimethylamine N-Oxide
M.K. Desmond and J.F. Siebenaller
Added KCl increases the apparent Michaelis constant (Km
) of pyruvate for porcine muscle-type lactate dehydrogenase
(100 mM KCl, 83%; 200 mM KCl, 188%). The effects of 100 mM
KCl were fully reversed by 375 mM trimethylamine N-oxide (TMAO).
TMAO (375-750 mM) partially reversed the effects of 200 mM
KCl. TMAO as the sole solute, at concentrations up to 750
mM, had no effect on Km. This is atypical because
compensatory osmolytes such as TMAO characteristically counteract
protein perturbation in an additive manner.
[Back to top]
Expression of Soluble and Functional Snake Venom
Fibrinolytic Enzyme Fibrolase Via the Co-Expression
of DsbC in Escherichia coli
S.-T. Zhang, J. Shi, J. Zhao, Y.-F. Qi and A.-G. Guo
Fibrolase is a non-hemorrhagic zinc metalloproteinase found
in southern copperhead snake venom (Agkistrodon contortrix
contortrix). It is capable of degrading fibrin clots
that result from purified fibrinogen or blood plasma. The
DNA of fibrolase was amplified by recursive PCR, and cloned
into the pET25b(+) expression vector. The effect of co-expression
of signalless versions of catalysts or molecular chaperones
FkpA, Skp and DsbC in cytoplasm was examined. When co-expressed
with DsbC, compared to the totally insoluble inclusion bodies
of fibrolase expressed separately, more than 90 % of recombinant
fibrolase was soluble, according to denaturing polyacrylamide
gel electrophoresis analysis. We also determined that FkpA
and Skp had no effects on the solubility of target protein
when co-expressed with fibrolase in Escherichia coli.
Fibrolase was successfully purified using metal ion affinity
chromatography and hydrophobic chromatography, and a maximum
yield of 20 mg/L fibrolase was obtained. Fibrinolytic activity
of recombinant fibrolase was demonstrated using fibrin plate
assays and fibrinogen hydrolysis.
[Back to top]
Kinetics of Inactivation of Phytase (phy A) During
Modification of Histidine Residue by IAA and DEP
X.-Y. Wang, M.-L. Sun, D.-M. Zhao and M. Wang
Chemical probing of histidine residues using specific modifiers,
iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted
in the inactivation of phytase (phy A). The kinetic theory
of the substrate reaction during the modification of enzyme
activity was applied to a study of the kinetics of the course
of inactivation of phytase by IAA and DEP. The results suggested
that histidine residues are involved in the active site of
the enzyme. They also indicated that inactivation of the enzyme
by IAA was via a complexing type inhibition, while the inhibition
by DEP reaction involved a conformational change step before
inactivation. The dissociation constant of the enzyme-inhibitor
complex of IAA, the constant of the conformational change
of DEP and the microscopic rate constants of two inhibitors
were obtained.
[Back to top]
cDNA Cloning, Purification and Properties of Paralithodes
camtschatica Metalloprotease
S.A. Semenova, G.N. Rudenskaya, D.V. Rebrikov and V.A.
Isaev
Hepatopancreas of king crab Paralithodes camtschatica
produces a metalloprotease, which belongs to the astacin family,
as cDNA cloning and sequencing showed. The metalloprotease
has been purified chromatographically to apparent homogeneity.
The purification factor was 16 and activity recovery was 20%.
pH and temperature optimum have been determined. In its properties
(molecular weight, pI, metal content) the metalloprotease
is close to crayfish astacin. However, analysis of the enzyme
sequences revealed differences, which account for differences
in substrate specificities and imply a different activation
mechanism.
[Back to top]
Juxtaposed Half-Cystines as Disulphide Bridged Partners
in Protein Tertiary Structure
K. Guruprasad, V.J. Kartik, T. Lavanya and L. Guruprasad
Disulphide bridges involving juxtaposed half-cystines
are observed in a number of protein three-dimensional structures
analyzed from the Protein Data Bank. These disulphide bridges
comprise a ‘ring of 8-atoms’ corresponding to
Cα1-C’-N Cα2-Cβ2-Sγ2-Sγ1-Cβ1-Cα1
in the two half-cystines. The presence of such disulphide
bridges introduces a ‘bend’ or ‘kink’
in the protein polypeptide chain.
[Back to top]
Spectroscopic Studies on Erythrina lysistemon
Seed Lectin Effect of Denaturing Agents on Lectin: Quaternary
Structure
E.H.E. Konozy, M.B. Dafalla and C.-D. Hsiao
The equilibrium unfolding of ElysL, a homodimeric legume lectin,
was studied using different denaturing agents such as guanidinium
chloride (GdnHCl), temperature and pH. Simultaneously, changes
in the secondary as well as tertiary structure of lectin were
followed by CD spectroscopy examination in both far and near-UV
region, respectively. The hydrophobic cluster binding dye,
1-anilino-8-naphthalene sulfonate (ANS), was used to further
explore intermediates and to follow the unfolding pathway
of lectin. The adenine binding ability of lectin was examined
and monitored via absorption spectra and the intrinsic
tryptophan fluorescence. Our findings indicate that the ElysL
unfolding process occurs via a three state pathway with an
intermediate state. We also showed that ElysL binds adenine
in a manner that involves a hydrophobic binding pocket that
is independent of the carbohydrate binding sites.
[Back to top]
Data Mining of VDJ Genes Reveals Interesting Clues
R.R. Joshi and V.K. Gupta
Hypervariability of the complementary determining regions
in characteristic structure of Immnoglobulins and the distinct,
cell-specific expressions of the genes coding for this important
class of proteins pose intriguing problems in experimental
and computational/informatics research requiring a special
approach different from those for the other proteins. We present
here an Average Linkage Hierarchical Clustering of
the Homosapien VDJ genes and the Immunoglobulin polypeptides
generated by them using special kind of data structures and
correlation matrices in place of the microarray data. The
results reveal interesting clues on the heterogeneity of exon
- intron locations in these gene-families and its possible
role in hypervariability of the Immunoglobulins.
[Back to top]
The Prediction of Hydrophobicity Gradients within
Transmembrane Protein α-Helices
Using a Novel Graphical Technique
F. Harris, S. Dennison and D.A. Phoenix
Here, it is shown that amphiphilicity profiling based on the
mean hydrophobic moment provides a simple visual guide for
the identification of oblique orientated α
-helices. The methodology has an efficiency of circa
70% and predicts that approximately 40% of transmembrane α
-helices may possess these structures.
[Back to top]
Determination of Mutation Trend in Hemagglutinins
by Means of Translation Probability Between RNA Codons and
Mutated Amino Acids
G. Wu and S. Yan
In this study, we used the 183 translation probabilities
between RNA codons and mutated amino acids to construct the
theoretical distributions of mutated amino acids in hemagglutinins
of influenza A virus. We then compared the actual distributions
of mutated amino acids from 953 hemagglutinins with their
theoretical ones. The results demonstrated that mutated amino
acids generally follow the direction of the theoretical distributions
governed by RNA codons. This, in turn, highlights the mutation
trend of amino acids in hemagglutinins and provides a method
for estimating possible mutations in a protein according to
its theoretical distributions of mutated amino acids.
[Back to top]
Efficient Expression of Haloarchaeal Nucleoside Diphosphate
Kinase Via Strong Porin Promoter in Moderately Halophilic
Bacteria
C. Nagayoshi, H. Tokunaga, A. Hayashi, H. Harazono, K.
Hamasaki, A. Ando and M. Tokunaga
Enzymes from extremely halophilic archaea require high concentration
of salts for their proper folding and consequently are expressed
as an unfolded and inactive form in Escherichia coli.
Moderate halophile, which accumulates protein stabilizers,
i.e., compatible solutes, is an attractive host cell for the
recombinant production of heterologous proteins, since such
protein stabilizers may help folding of expressed proteins.
Here, we succeeded in efficient expression and purification
to homogeneity of recombinant haloarchaeal nucleoside diphosphate
kinase (HsNDK) in moderate halophile using newly isolated
strong porin promoter.
[Back to top]
Characterization of Bovine Tau-Preparations by
Two-Dimensional Gel Electrophoresis
D. Volke and R. Hoffmann
The molecular diversity of bovine 
obtained by three different preparation protocols was characterized
by immunoblot analyses after two-dimensional gel electrophoresis.
These analyses revealed that
was heterogeneously modified, that is, up to 20 spots separated
along the pH gradient, mostly independent of the preparation
protocol.
[Back to top]
Crystallization and Preliminary X-Ray Analysis
of Sau3AI C-Terminal 233-419 Fragment
F. Yu, J. Song, C. Xu, Y. Ding, X. Hu, J. He and Z. Zhang
The C-terminal 232-419 amino acids fragment of endonuclease
Sau3AI has been successfully expressed in Escherichia
coli with 6 His at its N-terminal. After purification
and crystallization, one completed 2.8 Å data set was
collected using a Rigaku R-AXIS IV++ diffractometer.
The plate-like crystals belong to orthorhombic space group
P212121 with the cell dimension
of a = 34.75, b = 76.82, c = 123.59Å
and contain one molecule per asymmetric unit.
[Back to top]
Crystallization and Preliminary X-Ray Diffraction Analysis
of Three Mastoparans
F. Wang, X. Liu, H. Li, X. Lang, H. Peng, S. Liu and T.
Jiang
Mastoparans are tetradecapeptides found to be the major component
of wasp venoms. These peptides possess a variety of biological
activities. Three related mastoparans, mastoparan from Polistes
jadwagae (MP-PJ), mastoparanX (MP-X) and its carboxyl-free
C-terminal form (MP-X-COO-), were crystallized.
X-ray diffraction data for them were collected at resolutions
of 1.2 Å, 2.0 Å and 3.3 Å respectively.
[Back to top]
Zebrafish Caspase-3: Molecular Cloning, Characterization,
Crystallization and Phylogenetic Analysis
C. Chakraborty, S.S. Nandi, S. Sinha and V.K. Gera
Apoptosis plays an important role in maintaining the
normal function of various tissues and organs in different
species. Caspase-3 is a terminal caspases which plays an important
role in the execution of apoptosis in all vertebrates. It
was cloned from zebra fish embryos and its properties were
identified through Western blotting and biological activity.
In the cells over-expressing caspase-3, Western blotting with
an anti-His-tag antibody confirmed the presence of caspase-3
in the three bands that were proposed to correspond to the
precursor form (33 kDa), the mature forms processed at the
prodomain alone (29 kDa, large subunit) and small sub unit
(13 kD). Fish kidney cells were transiently co-transfected
with the β-galactosidase
reporter gene and either vector alone (mock), pZCASP3His (caspase-3)
or pZCASP3His mutant (caspase-3 mutant). After 72 h following
transfection of fish kidney cells, 35% of cells transfected
with the zebra fish caspase-3 construct, pZCASP3His, showed
apoptotic morphology when compared with cells transfected
with the mock vector or an expression construct (pZCASP3His
mutant) encoding the caspase-3 mutant lacking Cys. The fusion
proteins were expressed in Escherichia coli, isolated
from cell lysates by nickel-affinity column chromatography,
and cleaved with thrombin. A thrombin cleavage recognition
site was positioned at the fusion junction to release the
caspase-3 from the fusion protein. Phylogenetic analysis showed
that the cloned zebra fish caspase was a member of the caspase-3
subfamily with approximately 60% identity with caspase-3 from
Xenopus, chicken and mammals. We have obtained structural
information by X-ray crystallography. Orthorhombic crystals
of the caspase-3 that diffracted to 1.8 Å were obtained
in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH
7.0 –7.5), containing 30% glycerol. The space group
is C222 with cell dimensions of a = 36.07 Å,
b = 38.80 Å, c = 135.20 Å.
[Back to top]
Expression, Purification, Crystallization and
Preliminary Crystallographic Analysis of the Human Intracellular
Chloride Channel Protein CLIC4
D.-F. Li, Y.-F. Li, Q.-H. Huang, Y. Zhang and D.-C. Wang
The human chloride intracellular channel protein CLIC4
has been crystallized by the hanging-drop vapour-diffusion
technique using trisodium citrate as the precipitant. The
best crystals were obtained by the microseeding method. The
crystals diffracted to 2.2 Å resolution and were found
to belong to space group P121, with unit-cell parameters a
= 73.19, b =86.05, c = 73.38 Å, β
= 112.99° and three molecule per asymmetric unit.
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