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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 13, Number 4, 2006
Contents
Regular Papers
Unfolding During Urea Denaturation of a Low Molecular
Weight Phytocystatin (Thiol Protease Inhibitor) Purified from
Phaseolus mungo (Urd) Pp. 323-329
S. Sharma, F. Rashid and B. Bano
[Abstract]
Cyclization of α-Synuclein
Derived Peptide Increases Its Chaperone-Like Activity Pp.
331-333
T.D. Kim
[Abstract]
NMR Characterization of Recombinant Transmembrane
Protein CB2 Fragment CB2180-233
Pp. 335-342
J. Zhao, H. Zheng and X.-Q. Xie
[Abstract]
Production and Purification of Recombinant Human
Glucagon Overexpressed as Intein Fusion Protein in Escherichia
coli Pp. 343-347
R.S. Esipov, V.N. Stepanenko, A.I. Gurevich,
L.A. Chupova and A.I. Miroshnikov
[Abstract]
The Odorant-Binding Protein from Canis familiaris:
Purification, Characterization and New Perspectives in Biohazard
Assessment Pp. 349-352
S. D’Auria, M. Staiano, A. Varriale, V.
Scognamiglio, M. Rossi, A. Parracino, S. Campopiano, N. Cennamo
and L. Zeni
[Abstract]
Expression of Active α-N-Acetylgluco-saminidase/TAT
Chimerae in Cultured Spodoptera frugiperda Cells
Pp. 353-356
J.C. Bandsmer, T.N. Campbell, I. Cheyne and F.Y.M.
Choy
[Abstract]
Tyrosine Sulfation of Arylsulfatase A and Its
Peptide Pp. 357-361
C. Kasinathan, S. Jean and P. Manowitz
[Abstract]
Purification and Properties of a Bovine Uricase
Pp. 363-368
M.I. Rajoka, Khalil-ur-Rehman, M. Mehraj, M.W. Akhtar and
M.A. Zia
[Abstract]
Expression, Purification and Characterization
of an Enzymatically Active Truncated Human Rho-Kinase I (ROCK
I) Domain Expressed in Sf-9 Insect Cells Pp.
369-376
S.S. Khandekar, T. Yi, E. Dul, L.L. Wright, S.
Chen, G.F. Scott, G.K. Smith, D. Lee, E. Hu and R.B. Kirkpatrick
[Abstract]
Fate of Influenza A Virus Proteins Pp.
377-384
G. Wu and S. Yan
[Abstract]
The Early Events of α-Synuclein
Oligomerization Revealed by Photo-Induced Cross-Linking Pp.
385-390
H.-T. Li, X.-J. Lin, Y.-Y. Xie and H.-Y. Hu
[Abstract]
Network Analysis of the Protein Chain Tertiary
Structures of Heterocomplexes+ Pp.
391-396
J.-J. Li, D.-S. Huang, T.-M. Lok, M.R. Lyu, Y.-X.
Li and Y.-P. Zhu
[Abstract]
Leukocyte Function-Associated Antigen-1: Structure,
Function and Application Prospects Pp. 397-400
M. Xingyuan, Z. Wenyun and W. Tianwen
[Abstract]
Comparative Study of Apoptosis Induced
by H2O2
and NO: Limitation of Apoptosis Induced by NO Due to Slower
Recovery of Activity of NO-Modified Caspase Pp. 401-404
J.E. Kim, H. Seok, P. Karki, J.S. Lee, S.Y. Shin,
B. Cho and I.-S. Park
[Abstract]
Purification and Characterization of Arginine Kinase
from Locust Pp. 405-410
M. Li, X.-Y. Wang and J.-G. Bai
[Abstract]
Purification and Properties of Laticeptin, an Antimicrobial
Peptide from Skin Secretions of the South American Frog Leptodactylus
laticeps Pp. 411-415
J.M. Conlon, N. Al-Ghaferi, B. Abraham, A. Sonnevend,
J.D. King and P.F. Nielsen
[Abstract]
Crystallization Report
Crystallization and Preliminary X-Ray Crystallographic
Analysis of Yeast Tyrosyl-tRNA Synthetase Complexed with Its
Cognate tRNA Pp. 417-419
Y. Kusakabe, S. Ohno, N. Tanaka, M. Nakamura,
M. Tsunoda, T. Moriguchi, N. Asai, M. Sekine, T. Yokogawa,
K. Nishikawa and K.T. Nakamura
[Abstract]
Abstracts
[Back to top]
Unfolding During Urea Denaturation of a Low Molecular
Weight Phytocystatin (Thiol Protease Inhibitor) Purified from
Phaseolus mungo (Urd)
S. Sharma, F. Rashid and B. Bano
In the present study, two phytocystatins were purified
to homogeneity as peaks I and II with molecular weights of
19 kDa and 17 kDa, respectively, as determined by SDS-PAGE
and mass spectrometry. Both PMCs I and II were purified with
a greater than 1000-fold purification and overall yield of
about 16-18%. The effect of urea on PMC I and II was analysed
by fluorescence and Circular Dichroism (CD) spectroscopy.
Fluorescence studies suggest a red shift of the maximum emission
at higher urea concentrations. PMC I and II are extremely
stable protein inhibitors with regards to temperature and
pH stability. FTIR studies show predominant α-helical
structure in both the cystatins. CD analysis results show
change in urea concentration-dependent loss in ellipticity,
as well as in the shape of the CD spectrum compared to the
intact phytocystatin.
[Back to top]
Cyclization of α-Synuclein
Derived Peptide Increases Its Chaperone-Like Activity
T.D. Kim
We have analyzed a series of peptides derived from the C-terminus
of α-synuclein
for chaperone-like activity. Specifically, a cyclic peptide
generated by introducing a disulfide bond was observed to
increase chaperone-like activity. This is the first example
of a disulfide-crosslinked peptide that exhibits activity
against protein aggregation and activity loss.
[Back to top]
NMR Characterization of Recombinant Transmembrane
Protein CB2 Fragment CB2180-233
J. Zhao, H. Zheng and X.-Q. Xie
The expression of membrane proteins has been the bottleneck
for their structural studies. Recently, we developed a method
to obtain milligram quantities of isotope-labeled seven transmembrane
G-protein coupled cannabinoid (CB) receptor fragment in E.
coli. In order to verify this method and confirm the
recombinant isotope-labeled CB2 fragment, 3D hetero-nuclear
NMR techniques were used to analyze the structure of the fragment
CB2180-233
in DMSO-d6 solvent. The sequential assignments of TM5 and
intra-cellular loop 3 were accomplished, which confirmed the
experimental protocols of isotope-labeled recombinant protein
expression, fusion protein cleavage, and membrane protein
purification. The obtained structure also showed α-helix
in the TM5 region, but it was interrupted by a disordered
region (Gly204_ILe206). These results further revealed that
our established approach is a promising method to express
recombinant membrane proteins for their structural studies.
[Back to top]
Production and Purification of Recombinant Human Glucagon
Overexpressed as Intein Fusion Protein in Escherichia
coli
R.S. Esipov, V.N. Stepanenko, A.I. Gurevich,
L.A. Chupova and A.I. Miroshnikov
Chemico-enzymatic synthesis and cloning in Esherichia
coli of an artificial gene coding human glucagon was
performed. Recombinant plasmid containing hybrid glucagons
gene and intein Ssp dnaB from Synechocestis sp.
was designed. Expression of the obtained hybrid gene in E.
coli, properties of the formed hybrid protein, and conditions
of its autocatalytic cleavage leading to glucagon formation
were studied.
[Back to top]
The Odorant-Binding Protein from Canis familiaris:
Purification, Characterization and New Perspectives in Biohazard
Assessment
S. D’Auria, M. Staiano, A. Varriale, V.
Scognamiglio, M. Rossi, A. Parracino, S. Campopiano, N. Cennamo
and L. Zeni
In this report we show the purification to homogeneity and
a partial characterization of a new odorant-binding protein
from Canis familiar (CfOBP) nasal mucosa.
In addition, we report preliminary data on the utilization
of CfOBP as a probe for the development of a refractive
index-based biosensor.
[Back to top]
Expression of Active α-N-Acetylgluco-saminidase/TAT
Chimerae in Cultured Spodoptera frugiperda Cells
J.C. Bandsmer, T.N. Campbell, I. Cheyne and F.Y.M.
Choy
We examined the production and secretion of fusion constructs
containing α-N-acetylglucosaminidase,
the enzyme deficient in Sanfilippo B, and either wildtype
TAT or modified TAT in cultured Spodoptera frugiperda
cells. All constructs exhibited successful expression of active
enzyme, suggesting the future possibility of utilizing TAT/α-N-acetylglucosaminidase
chimerae in enzyme replacement therapy.
[Back to top]
Tyrosine Sulfation of Arylsulfatase A and Its Peptide
C. Kasinathan, S. Jean and P. Manowitz
Purified human liver arylsulfatase A (ASA) as well as an
ASA peptide (residues 28-39) were sulfated by tyrosyl protein
sulfotransferase in vitro. The media, but not the
cell lysate, of normal human fibroblasts contained a tyrosine
sulfated protein (pI = 4.5-5.5). This protein was not present
in either media or cell lysate of human fibroblasts lacking
ASA protein. These results suggest that tyrosine sulfation
facilitates secretion of ASA and that this may have pathophysiological
consequences.
[Back to top]
Purification and Properties of a Bovine Uricase
M.I. Rajoka, Khalil-ur-Rehman, M. Mehraj, M.W. Akhtar and
M.A. Zia
Uricase from bovine kidney, purified to homogeneity level,
had a molecular weight of 70 kDa. The apparent Km
and Vmax
values for uric acid hydrolysis were 0.125 mM and 102 IU mg-1
protein respectively. The activation energy requirement for
uric acid hydrolysis by uricase and inactivation of enzyme
were 11.6 and 14.5 kJ/M respectively. Both enthalpy Δ
H*) and entropy of activation (Δ S*) for uricase activity
were lower than those reported for some thermostable enzymes.
[Back to top]
Expression, Purification and Characterization of an
Enzymatically Active Truncated Human Rho-Kinase I (ROCK I)
Domain Expressed in Sf-9 Insect Cells
S.S. Khandekar, T. Yi, E. Dul, L.L. Wright, S.
Chen, G.F. Scott, G.K. Smith, D. Lee, E. Hu and R.B. Kirkpatrick
Rho Kinase I (ROCK I) is a serine/threonine kinase that is
involved in diverse cellular signaling. To further understand
the physiological role of ROCK I and to identify and develop
potent and selective inhibitors of ROCK I, we have overexpressed
and purified a constitutively active dimeric human ROCK I
(3-543) kinase domain using the Sf9-baculovirus expression
system. In addition, using a limited proteolysis technique,
we have identified a minimal functional subdomain of ROCK
I that can be used in crystallization studies. The availability
of multimilligram amounts of purified and well characterized
functional human ROCK I kinase domains will be useful in screening
and structural studies.
[Back to top]
Fate of Influenza A Virus Proteins
G. Wu and S. Yan
In this review we summarize the current state, history, future,
mutation tendency and species susceptibility of influenza
A virus proteins based on our probabilistic analyses on amino
acid pairs, and compare the current state of influenza A virus
proteins with that of proteins which we have studied in the
past.
[Back to top]
The Early Events of α-Synuclein
Oligomerization Revealed by Photo-Induced Cross-Linking
H.-T. Li, X.-J. Lin, Y.-Y. Xie and H.-Y. Hu
Assembly of α-synuclein
(α-Syn)
into neurotoxic oligomers and fibrils is an important pathogenic
feature of Parkinson’s disease. Studying the early events
of α-Syn
aggregation, such as oligomerization and nucleation, is indispensable
to understanding the complicated process. Here, photo-induced
cross-linking of unmodified proteins (PICUP) technique is
applied to elucidate the early-stage oligomerization of α-Syn.
Results show that α-Syn
in solution exhibits a mixture of various species, including
at least monomers, dimers and trimers. Aggregation of α-Syn
probably originates from the dimeric and trimeric seeds. Furthermore,
the N-terminal amphipathic region is proposed to be required
for the oli-gomerization (dimerization and trimerization)
process. This observation may extend our knowledge on the
early events of α-Syn
aggregation and the neurotoxic aggregation species.
[Back to top]
Network Analysis of the Protein Chain Tertiary Structures
of Heterocomplexes+
J.-J. Li, D.-S. Huang, T.-M. Lok, M.R. Lyu, Y.-X.
Li and Y.-P. Zhu
In this paper, the tertiary structures of protein chains
of heterocomplexes were mapped to 2D networks; based on the
mapping approach, statistical properties of these networks
were systematically studied. Firstly, our experimental results
confirmed that the networks derived from protein structures
possess small-world properties. Secondly, an interesting relationship
between network average degree and the network size was discovered,
which was quantified as an empirical function enabling us
to estimate the number of residue contacts of the protein
chains accurately. Thirdly, by analyzing the average clustering
coefficient for nodes having the same degree in the network,
it was found that the architectures of the networks and protein
structures analyzed are hierarchically organized. Finally,
network motifs were detected in the networks which are believed
to determine the family or superfamily the networks belong
to. The study of protein structures with the new perspective
might shed some light on understanding the underlying laws
of evolution, function and structures of proteins, and therefore
would be complementary to other currently existing methods.
[Back to top]
Leukocyte Function-Associated Antigen-1: Structure,
Function and Application Prospects
M. Xingyuan, Z. Wenyun and W. Tianwen
This review focuses on the structure, function and pathological
role of leukocyte function-associated antigen-1 (LFA-1) which
is a heterodimeric protein consisting of two subunits. LFA-1
plays a most important role in the immune system including
adhesion, extravasation, migration, apoptosis, cytotoxicity,
cytokine production, and proliferation of lymphocytes. Therefore,
T-cell activation can be suppressed by blocking ICAM-1/LFA-1
interaction in autoimmune diseases and organ transplantation.
Many different inhibitors (i.e. antibodies, peptides, small
molecules) have been demonstrated to block ICAM-1/LFA-1 interaction,
and some of them are promising for medical treatment or have
reached clinical trials.
[Back to top]
Comparative Study of Apoptosis Induced by H2O2
and NO: Limitation of Apoptosis Induced by NO Due to Slower
Recovery of Activity of NO-Modified Caspase
J.E. Kim, H. Seok, P. Karki, J.S. Lee, S.Y. Shin,
B. Cho and I.-S. Park
Both H2O2
and NO can act as apoptogens, triggering apoptosis in many
cells. They are also well known inhibitors of caspases, essential
enzymes in apoptosis. The differences between these two agents
as apoptosis inducers and how caspases mediate apoptosis with
these inhibitory agents is still unclear. Consistent with
the previous reports, these two agents induced apoptosis accompanied
by caspase activation with limitation of all apoptotic events
for NO. It was found that NO-modified caspase-3 showed a slower
recovery of its activity in the presence of the reducing agents
compared to that of H2O2
modification. This is one possible cause of the limited apoptosis
in the case of NO.
[Back to top]
Purification and Characterization of Arginine
Kinase from Locust
M. Li, X.-Y. Wang and J.-G. Bai
L-Arginine kinase (AK; ATP:L-arginine N-phosphotransferase;
EC 2.7.3.3) catalyzes the reversible transphosphorylation
between N-phospho-L-arginine (PArg) and ATP thus
buffering cellular ATP levels. AK was purified from the leg
muscle of the locust Migratoria manilensis by Sephacryl
S-200 HR gel filtration chromatography and DEAE Sepharose
CL-6B fast flow anion exchange chromatography to an apparent
homogeneity with a recovery of 80%. The enzyme behaved as
monomeric protein with molecular mass of about 40 kD, and
had a pH and temperature optimum of 8.6 and 30°C,
respectively, and a pI of about 6.3. The Michaelis constants
for synthesis of PArg are 0.936 and 1.290 mM for L-arginine
and ATP, respectively and kcat/KmArg
174. The activity of AK required divalent cations such as
Mg2+ and Mn2+. In the presence of Cu2+
and Zn2+, AK activity was greatly inhibited. The
intrinsic protein fluorescence emission maximum at 330 nm
using the excitation wavelength at 295 nm suggested that tryptophan
residues are below the surface of the protein and not exposed
to solvent.
[Back to top]
Purification and Properties of Laticeptin, an Antimicrobial
Peptide from Skin Secretions of the South American Frog Leptodactylus
laticeps
J.M. Conlon, N. Al-Ghaferi, B. Abraham, A. Sonnevend,
J.D. King and P.F. Nielsen
Norepinephrine-stimulated skin secretions from the Sante
Fe frog Leptodactylus laticeps contained high concentrations
of a peptide, termed laticeptin, with the primary structure
Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Leu-Ala-Gly-His-Leu-Ala-Thr-Lys-Val-Met-Asn-Lys-Leu.NH2.
Laticeptin inhibited the growth of selected Gram-negative
bacteria but the lack of activity against Gram-positive bacteria
and the very low hemolytic activity is probably a consequence
of the weak amphipathicity of the peptide in its α-helical
conformation.
[Back to top]
Crystallization and Preliminary X-Ray Crystallographic
Analysis of Yeast Tyrosyl-tRNA Synthetase Complexed with Its
Cognate tRNA
Y. Kusakabe, S. Ohno, N. Tanaka, M. Nakamura,
M. Tsunoda, T. Moriguchi, N. Asai, M. Sekine, T. Yokogawa,
K. Nishikawa and K.T. Nakamura
Yeast tyrosyl-tRNA synthetase (yTyrRS) has been crystallized
by the vapor diffusion method in the presence of its cognate
tRNATyr. The crystals belong to a tetragonal
space group P41212
with cell dimensions of a = b = 63.85 Å,
and c = 330.3 Å. The asymmetric unit contains
one molecule each of yTyrRS and tRNATyr
(one-half of a 2:2 complex). X-ray diffraction data have been
collected up to 2.5 Å resolution.
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