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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 15 Number 1, 2008
Contents
Solid Phase Synthesis of a Glycopeptide Analogue Using
the Acid Sensitive 4-Methoxybenzhydryl Bromide Resin
Pp. 1-5
T. Tselios, K. Kelaidonis, A. Resvani, K. Prousalis, J.
Matsoukas and T. Tsegenidis
[Abstract]
Determination of Some Kinetic and Characteristic Properties
of Glutathione S-transferase from Bovine Erythrocytes
Pp. 6-12
S. Güvercin, M. Erat and H. Sakiroglu
[Abstract]
DNA Cleavage Study Using Copper (II)-GlyAibHis: A
Tripeptide Complex Based on ATCUN Peptide Motifs
Pp. 13-9
R.K. Singh, N.K. Sharma, R. Prasad and U.P. Singh
[Abstract]
Oxidative Stress Induced Functional and Structural
Modifications of High Molecular Mass Goat Brain Cystatin
Pp. 20-26
S. Sumbul and B. Bano
[Abstract]
Molecular Modeling of Two CYP2C19 SNPs and Its
Implications for Personalized Drug Design Pp. 27-32
J.-F. Wang, D.-Q. Wei, C. Chen, Y. Li and K.-C. Chou
[Abstract]
COILCHECK: An Interactive Server for the Analysis
of Interface Regions in Coiled Coils Pp. 33-38
V. Alva, D.P.S. Devi and R. Sowdhamini
[Abstract]
Effect of Ca2+
on Beta-Propeller Phytases Pp. 39-42
S. Fu, J. Sun and L. Qian
[Abstract]
Detection of a γ-Carboxy-Glutamate
as Novel Post Translational Modification of Human Transthyretin
Pp. 43-46
S. Rüggeberg, P. Horn, X. Li, P. Vajkoczy and T.
Franz
[Abstract]
The Heavy-Light Chain Loop of Human Cathepsin-L Modulates
Its Activity and Stability Pp. 47-53
M. Fairhead and C.F. van der Walle
[Abstract]
The Hypervariable D3 Domain of Salmonella Flagellin
Is an Autonomous Folding Unit Pp. 54-57
A. Sebestyén, A. Muskotál, B.M. Végh
and F. Vonderviszt
[Abstract]
S-adenosylmethionine Inhibits Ubiquitin-Proteasome
System In Vitro and on Rat Vascular Smooth Muscle
Cells Pp. 58-62
F. D’Anselmi, A. Cucina, G. Cavallaro, M. Bizzarri,
R.A. Cavallaro, A. Fuso, A. Cavallaro and S. Scarpa
[Abstract]
Secondary Structure of the MiRP1 (KCNE2) Potassium
Channel Ancillary Subunit Pp. 63-75
G.W. Abbott, B. Ramesh and S.K.S. Srai
[Abstract]
The Interaction Between Two Arabidopsis Polyadenylation Factor
Subunits Involves an Evolutionarily-Conserved Motif and Has
Implications for the Assembly and Function of the Polyadenylation
Complex Pp. 76-88
B. Addepalli and A.G. Hunt
[Abstract]
Processing of DNA Replication and Repair Intermediates
by the Concerted Action of RecQ Helicases and Rad2 Structure-Specific
Nucleases Pp. 89-102
S. Sharma, J.A. Sommers and R.M. Brosh, Jr.
[Abstract]
Interaction of Calreticulin with Amyloid Beta
Peptide 1-42 Pp. 103-107
K. Duus, P.R. Hansen and G. Houen
[Abstract]
Structural and Functional Diversity of Nudix
Fold Pp. 108-112
J. Lin, B. Tian and Y. Hua
[Abstract]
Crystallization Reports
Crystallization and Preliminary X-Ray Analysis of
Fluorescent Protein mBanana Pp. 113-114
Y. Zhou, Y. Wu, J. Song, Y. Ding, X. Hu and Z. Zhang
[Abstract]
Purification, Crystallization and Preliminary
X-Ray Studies of AxCesD Required for Efficient Cellulose Biosynthesis
in Acetobacter xylinum Pp. 115-117
S.Q. Hu, Y.G. Gao, K. Tajima, M. Yao, T. Yoda, D. Shimura,
Y. Satoh, S. Kawano, I. Tanaka and M. Munekata
[Abstract]
Abstracts
[Back to top]
Solid Phase Synthesis of a Glycopeptide Analogue Using
the Acid Sensitive 4-Methoxybenzhydryl Bromide Resin
T. Tselios, K. Kelaidonis, A. Resvani, K. Prousalis, J.
Matsoukas and T. Tsegenidis
A convenient solid phase synthesis of a Thrombin Receptor
Glycopeptide Mimetic analogue namely, 1-O-Methyl-2-N-{1´-(argininocarbonyl)-4´-[(4´´-fluoro)-benzylamido]-cyclohexane}-glucosamine
using Fmoc/tBu methodology and the 4-Methoxybenzhydryl bromide
resin is described. The synthesized analogue was purified
by Reverse Phase High Performance Liquid Chromatography (RP-HPLC)
and was identified by Electron Spray Ionization-Mass Spectrometry
(ESI-MS) and Nuclear Magnetic Resonance (NMR). The synthetic
protocol introduced for the first time successfully the acid
sensitive 4-Methoxybenzhydryl bromide resin as a scaffold
for the synthesis of glycopeptides resulting in high yield
reactions. This synthetic procedure could be a general one
for the convenient synthesis of such glyco compounds as the
method was used for the first time to glycosylate a non peptide
mimetic of an important protein sequence, in particular of
the thrombin receptor active site S42FLLR46.
[Back to top]
Determination of Some Kinetic and Characteristic Properties
of Glutathione S-transferase from Bovine Erythrocytes
S. Güvercin, M. Erat and H. Sakiroglu
Glutathione S-transferase was purified from bovine erythrocytes
and some kinetic and characteristic properties of the enzyme
were investigated. The purification procedure was composed
of preparation of homogenate and Glutathione-Agarose affinity
chromatography. Thanks to the procedure, the enzyme was purified
6,800 fold with 97% yield and a specific activity of 136 EU/mg
proteins. On sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS/PAGE), one band with a mass of 27 kDa was found. The
native molecular weight of the enzyme was found to be approximately
53 kDa by Sephadex G-100 gel filtration chromatography. Optimum
pH, stable pH, optimum temperature, and optimum ionic strength
were determined as 7.0, 6.5 in K-phosphate buffer, 20 oC,
0.1 M K-phosphate, respectively. The best activity was obtained
with 1-chloro-2,4-dinitrobenzene (CDNB) in a study performed
with different substrates. Vmax,
Km, and kcat
values were calculated as 402.63 ± 4.99 EU/mg proteins,
0.7447 ± 0.0007 mM, and 11436 min-1
for CDNB, and 88.00 ± 2.30 EU/mg proteins, 0.3257 ±
0.0012 mM, and 477 min-1
for GSH, respectively, by using Lineweaver-Burk graphs obtained
from 1/V versus 1/[CDNB] and 1/[GSH].
[Back to top]
DNA Cleavage Study Using Copper (II)-GlyAibHis: A
Tripeptide Complex Based on ATCUN Peptide Motifs
R.K. Singh, N.K. Sharma, R. Prasad and U.P. Singh
Development of new chemical nucleases is a matter of
great interest because of their extensive use in biotechnology
and as therapeutic agents. The ATCUN (amino terminal Cu(II)
and Ni(II) binding) is a peptide motif that occurs naturally
in the serum albumins. The similar peptide motif (GlyAibHis)
having unnatural amino acid Aib (α-aminoisobutyric
acid) was synthesized and its Cu(II) complex was characterized
by ESI-MS and spectrophotometry studies. The reactivity of
this complex toward DNA cleavage has been investigated. Cu(II)-GlyAibHis
shows the DNA cleavage only in presence of mild oxidizing
agents like ascorbate by an oxidative mechanism rather than
hydrolytic and follows the pseudo first order kinetics (Kobs
= 0.085 min-1). The non-hydrolytic
mechanism was further supported by the hydrolysis of pNPP
which followed the pseudo first order kinetics (Kobs
= 1.98 x 10-2 min-1)
having no pH effect.
[Back to top]
Oxidative Stress Induced Functional and Structural
Modifications of High Molecular Mass Goat Brain Cystatin
S. Sumbul and B. Bano
Cystatins are thiol proteinase inhibitors ubiquitously
present in mammalian body. In brain, they prevent unwanted
proteolysis and are involved in several neurodegenerative
diseases. In the present study, it has been demonstrated that
photoactivated riboflavin and H2
O2 induced modifications
of high molecular mass goat brain cystatin (HM-GBC) leads
to its inactivation and degradation. It was found that the
damage with both the oxidants occurred mainly because of the
hydroxyl radicals. It has been also proposed that susceptibility
of HM-GBC to oxidation by reactive oxygen species generated
in vivo arise from oxidative modifications may lead
to damage of this significant protein as it is so well pro-nounced,
in vitro.
[Back to top]
Molecular Modeling of Two CYP2C19 SNPs and Its
Implications for Personalized Drug Design
J.-F. Wang, D.-Q. Wei, C. Chen, Y. Li and K.-C. Chou
CYP2C19 is an important member of the cytochrome
P-450 enzyme superfamily and plays a significant role in the
drug metabolism. In order to gain insights for developing
personalized drugs, the structure-activity relationships of
two SNPs, W120R and I331V, with the ligands of CEC, Fluvoxamine,
Lescol and Ticlopidine were investigated through the structure-activity
relationship approach. By means of a series of docking studies,
the binding pockets of the two SNPs for the four compounds
are explicitly defined that will be very useful for conducting
mutagenesis studies, providing insights into personalization
of drug treatments and stimulating novel strategies for finding
desired personalized drugs.
[Back to top]
COILCHECK: An Interactive Server for the Analysis
of Interface Regions in Coiled Coils
V. Alva, D.P.S. Devi and R. Sowdhamini
Coiled coils are important structural modules and
domains of protein-protein interactions. Further, they provide
the required framework to proteins, like kinesins, myosins
and SNAREs, which are either structural or involved in transport
of biomolecules. We provide an interactive webserver to measure
the strength of interactions between two helices involved
in coiled coils. Interactions are measured using non-bonded
and electrostatic interactions and the presence of hydrogen
bonds and salt bridges. The sum of these interactions is expressed
as psuedoenergy, whose ranges have been standardized using
all structural entries that are classified to contain coiled
coils. The results are displayed conveniently as energy per
residue along with options to obtain detailed list of different
types of interactions. This webserver can be useful to assess
the strength of coiled coil regions, to recognize weak and
strong regions, to rationalize the phenotypic behaviour of
single residue mutations as well as to design mutation experiments.
COILCHECK webserver can be accessed from http://caps.ncbs.res.in/coilcheck/
[Back to top]
Effect of Ca2+
on Beta-Propeller Phytases
S. Fu, J. Sun and L. Qian
Beta-propeller phytases (BPPs) are a special class
of enzyme that are mainly isolated from Bacillus
and are widely used in animal nutrition, human health and
environmental protection. BPPs class exhibits both unique
Ca2+-dependent catalytic
property and highly strict substrate specificity for the calcium-phytate
complex. This review describes the effect of Ca2+
on the catalytic activity, thermal stability, and structural
conformation of BPPs.
[Back to top]
Detection of a γ-Carboxy-Glutamate
as Novel Post Translational Modification of Human Transthyretin
S. Rüggeberg, P. Horn, X. Li, P. Vajkoczy and T.
Franz
During analysis of the proteome in the cerebrospinal fluid
(CSF) of the Caucasian form of moyamoya disease (MMD), a novel
post-translational modification of human transthyretin was
observed. Two-dimensional electrophoresis and subsequent peptide
sequencing with ESI-MS/MS were performed to discover the γ-carboxylation
of the Glu-42 (Gla-42).
[Back to top]
The Heavy-Light Chain Loop of Human Cathepsin-L Modulates
Its Activity and Stability
M. Fairhead and C.F. van der Walle
Differences evident in the sequence alignment of human
cathepsin-L with shrimp cathepsin-L and silicatein-α
suggest the indirect involvement of the heavy to light chain
loop (E286 to E289)
in the function of these enzymes. Deletion of the loop and
adjacent residues S290 to
N293, decreased specific
protease activity by 81% and 63%, respectively; complete substitution
for the corresponding silicatein-α
loop decreased activity by 35%. In all cases the Km was largely
unchanged. The conformational stability of human procathepsin-L
was not altered by deletion of E286
to E289 but increased on
deletion of S290 to N293.
Therefore, shortening the loop does not change substrate affinity
but does influence activity, in part via conformational change.
[Back to top]
The Hypervariable D3 Domain of Salmonella Flagellin
Is an Autonomous Folding Unit
A. Sebestyén, A. Muskotál, B.M. Végh
and F. Vonderviszt
The hypervariable D3 domain of Salmonella flagellin,
composed of the 190-285 segment, is the major determinant
of flagellar antigenicity. D3 was cloned and overexpressed
in E. coli. Although previous studies concluded that
D3 is stabilized by interactions with the D2 domain, our calorimetric
experiments have revealed that isolated D3 has a stable tertiary
structure which is highly resistant against proteolytic digestion.
Repeated heating experiments demonstrated that unfolding of
D3 is reversible. Its small size and stable structure makes
D3 a promising protein scaffold for the development of artificial
binding proteins by directed evolution.
[Back to top]
S-adenosylmethionine Inhibits Ubiquitin-Proteasome
System In Vitro and on Rat Vascular Smooth Muscle
Cells
F. D’Anselmi, A. Cucina, G. Cavallaro, M. Bizzarri,
R.A. Cavallaro, A. Fuso, A. Cavallaro and S. Scarpa
S-adenosylmethionine is a metabolite regulating many biological
processes; S-adenosylmethionine effect on ubiquitin-proteasome
system (UPS) has not been studied yet. We investigated S-adenosylmethionine
effects on UPS activity both in vitro, by inhibitor
screening assay, and in rat vascular smooth muscle cells,
by Western Blot of proteasomal targets. We found that S-adenosylmethionine
inhibited UPS activity.
[Back to top]
Secondary Structure of the MiRP1 (KCNE2) Potassium
Channel Ancillary Subunit
G.W. Abbott, B. Ramesh and S.K.S. Srai
MiRP1 (encoded by the KCNE2 gene) is one of a family
of five single transmembrane domain voltage-gated potassium
(Kv) channel ancillary subunits currently under intense scrutiny
to establish their position in channel complexes and elucidate
α subunit
contact points, but its structure is unknown. MiRP1 mutations
are associated with inherited and acquired cardiac arrhythmia.
Here, synthetic peptides corresponding to human MiRP1 (full-length
and separate domains) were structurally analyzed using FTIR
and CD spectroscopy. The N-terminal (extracellular) domain
was soluble and pre-dominantly non-ordered in aqueous media,
but predominantly α-helical
in L-α-lysophosphatidylcholine
(LPC) micelles. The MiRP1 transmembrane domain was predominantly
a mixture of α-helix
and non-ordered structure in LPC micelles, with a minor contribution
from non-aggregated β-strand.
The intracellular C-terminal domain was insoluble in aqueous
solution; reconstitution into non-aqueous environments resulted
in solubility and adoption of increasing amounts of α-helix,
with the solvent order sodium dodecyl sulphate < dimyristoyl
L-α-phosphatidylcholine
(DMPC) < LPC < trifluoroethanol. Correlation of secondary
structure changes with lipid transition temperature during
heating suggested that the MiRP1 C-terminus incorporates into
DMPC bilayers. Full-length MiRP1 was soluble in SDS micelles
and calculated to contain 34% α-helix,
23% β-strand
and 43% non-ordered structure in this environment, as determined
by CD spectroscopy.
Thus, MiRP1 is highly dependent upon hydrophobic interaction
via lipid and/or protein contacts for adoption of ordered
structure without nonspecific aggregation, consistent with
a role as a membrane-spanning subunit within Kv channel complexes.
These data will provide a structural framework for ongoing
mutagenesis-based in situ structure-function studies
of MiRP1 and its relatives.
[Back to top]
The Interaction Between Two Arabidopsis Polyadenylation Factor
Subunits Involves an Evolutionarily-Conserved Motif and Has
Implications for the Assembly and Function of the Polyadenylation
Complex
B. Addepalli and A.G. Hunt
The polyadenylation factor subunit “Factor Interacting
with Poly(A) polymerase” (Fip1) is an important bridging
subunit in the eukaryotic polyadenylation complex. To better
understand the functioning of Fip1 in Arabidopsis,
a random combinatorial screen for peptides that interact with
a conserved plant-specific domain in the protein was conducted.
A search of the Arabidopsis proteome using these
Fip1-binding peptides as queries resulted in the identification
of a number of putative Fip1-interacting proteins. One of
these was the polyadenylation factor subunit, CstF77. This
purported interaction was confirmed by yeast two-hybrid and
in vitro assays. Mutation of the motif identified
in the phage display screen eliminated the interaction, corroborating
the results of the phage display screen. The domain of CstF77
that inter-acts with Fip1 lies at its extreme C-terminus and
is distinct from the part of CstF77 that binds CstF64. Taken
together, these results suggest that Fip1 is situated near
CstF64 in the polyadenylation complex.
[Back to top]
Processing of DNA Replication and Repair Intermediates
by the Concerted Action of RecQ Helicases and Rad2 Structure-Specific
Nucleases
S. Sharma, J.A. Sommers and R.M. Brosh, Jr.
Processing of DNA replication and repair intermediates
is a critical aspect of genome stability maintenance. The
coordinated action of RecQ-like helicases with structure-specific
nucleases such as Flap Endonuclease 1 plays an important role
in the processing of certain DNA structures associated with
the replication fork, DNA repair, or telomeres. We will summarize
our current understanding of how and in what context these
interactions take place, with a particular emphasis on the
mechanisms of RecQ helicases in processing of key DNA replication
and repair intermediates by their protein interactions with
FEN-1 and related structure-specific nucleases.
[Back to top]
Interaction of Calreticulin with Amyloid Beta
Peptide 1-42
K. Duus, P.R. Hansen and G. Houen
The interaction of calreticulin with amyloid beta (Aβ)
was investigated using solid phase and solution binding assays.
Calreticulin bound Aβ
1-42 in a time and concentration dependent fashion. The binding
was optimal at pH 5 and was stimulated by Ca2+
and inhibited by Zn2+ at
pH 7. Interaction took place through the hydrophobic C-terminus
of Aβ
1-42 and the polypeptide binding site of calreticulin. The
results are discussed in the light of a reported role of calreticulin
as a cell surface scavenger receptor.
[Back to top]
Structural and Functional Diversity of Nudix
Fold
J. Lin, B. Tian and Y. Hua
Nudix hydrolases catalyze the hydrolysis of nucleoside
diphosphates linked to other moieties X, and contain the sequence
motif or Nudix box, GX5EX7REUXEEXGU. The mechanisms of Nudix
hydrolases are highly diverse in the position on the substrate.
In this paper, we examined the sequences and structures of
the MutT/Nudix superfamily. And two recent developed methods
were employed for data analyses of the superfamily. One is
QH method evaluating the similarities among structures for
structural phylogeny. The other method is clustering analysis
by using the CLANS program that could analysis thousands of
sequences of the full dataset rather than the representative
sequences of the superfamily. Finally, we proposed a more
objective classification of the MutT/Nudix superfamily members
based on detailed sequence and structure analyses.
[Back to top]
Crystallization and Preliminary X-Ray Analysis of
Fluorescent Protein mBanana
Y. Zhou, Y. Wu, J. Song, Y. Ding, X. Hu and Z. Zhang
mBanana is a novel monomeric red fluorescent protein
mutant. It was cloned and expressed in Escherichia coli
with 10 histidine residues at its N-terminal. After cleavage
of the His tag by TEV protease, the mBanana was further purified
and crystallized by the hanging-drop vapor-diffusion technique.
The crystals can diffract to 2.0Å resolution and one
set of completed data was collected. It showed that the orthorhombic
mBanana crystal was in space group P21 with unit cell parameters
(48.629, 42.667, 61.714, 90, 111.676, 90) and contained one
molecule in one asymmetric unit.
[Back to top]
Purification, Crystallization and Preliminary
X-Ray Studies of AxCesD Required for Efficient Cellulose Biosynthesis
in Acetobacter xylinum
S.Q. Hu, Y.G. Gao, K. Tajima, M. Yao, T. Yoda, D. Shimura,
Y. Satoh, S. Kawano, I. Tanaka and M. Munekata
AxCesD protein required for bacterial cellulose biosynthesis
in Acetobacter xylinum was overexpressed in E.
coli, purified and crystallized. Single crystals of SeMet-substituted
AxCesD were obtained by the sitting-drop vapor-diffusion method.
The crystal belongs to the primitive trigonal space group
P32, with unit-cell
parameters a = b = 77.7 Å, and c = 213.9 Å.
The asymmetric unit in the crystal was assumed to contain
8 protein molecules giving the Matthews coefficient (VM
) of 2.54 Å3 Da-1.
Se-MAD data were collected to 2.3 Å resolution using
synchrotron radiations.
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