|
Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 13, Number 8, 2006
Contents
Regular Papers
Purification and Structural Characterization of Human
ERp29 Pp. 753-759
J. Zheng, X. Liu, X. Yan, L. Dai and C. Ji
[Abstract]
A Simple Search of TM Segments in Polytopic Membrane
Protein Using Matrix-Assisted Laser Desorption Ionization
Time-of-Flight Mass Spectrometry Pp. 761-767
Y. Abe, T. Hamasaki, S. Turusaki, S. Takazaki, J. Xiui,
D. Kang and N. Hamasaki
[Abstract]
The Stability Curve of Hen Egg White Lysozyme
Pp. 769-772
S.S. Younvanich and B.M. Britt
[Abstract]
Quantitative Analysis of Cytochrome C Released
from Rice Mitochondria Using the Adsorptive Polarographic
Wave of Guanidine Modified Co(II)-Cytochrome C Complex Pp.
773-777
C. Xia, X. Liu, D. Luo, H. Wang, F. He, L. Yuan and C.
Wang
[Abstract]
Isolation and Partial Characterization of Ribonuclease
Inhibitor from Goat Liver Pp. 779-783
J. Chatterjee, T.K. Maiti and S. Dasgupta
[Abstract]
High-Level Expression and Purification of Human
Epidermal Growth Factor with SUMO Fusion in Escherichia
coli Pp. 785-792
Z. Su, Y. Huang, Q. Zhou, Z. Wu, X. Wu, Q. Zheng,
C. Ding and X. Li
[Abstract]
Effects of Physical, Ionic, and Structural Factors
on the Binding of Repressor of Mycobacteriophage L1 to Its
Cognate Operator DNA Pp. 793-798
T. Ganguly, P.K. Chanda, A. Bandhu, P. Chattoraj,
M. Das and S. Sau
[Abstract]
Purification and Thermal Characterization of
a Novel Peroxidase from a Local Chick Pea Cultivar Pp.
799-804
H.N. Bhatti, A. Najma, M. Asgher, M.A. Hanif
and M.A. Zia
[Abstract]
Antimicrobial Properties of Nuclear Diffusion
Inhibitory Signal of HIV-1 Rev Pp. 805-806
N. Kobayashi and T. Yoshida
[Abstract]
In Silico Criterion for Prediction of
Effects of p53 Gene Missense Mutations on p53-Mdm2 Feedback
Loop Pp. 807-814
N. Veljkovic and V. Perovic
[Abstract]
N-Terminal Mutational Analysis of the Interaction
Between Growth-Blocking Peptide (GBP) and Receptor of Insect
Immune Cells Pp. 815-822
S. Watanabe, M. Tada, T. Aizawa, M. Yoshida, T. Sugaya,
M. Taguchi, T. Kouno, T. Nakamura, M. Mizuguchi, M. Demura,
Y. Hayakawa and K. Kawano
[Abstract]
Expression and Purification of Exendin-4 Dimer
in Escherichia coli and Its Interaction with GLP-1
Receptor In Vitro Pp. 823-827
L. Yi, X. Yin, D. Wei and Y. Ma
[Abstract]
Transformation of a Biologically Active Peptide
into Peptoid Analogs While Retaining Biological Activity Pp.
829-833
B. Hoffmann, T. Ast, T. Polakowski, U. Reineke
and R. Volkmer
[Abstract]
Allergenic Tropomyosins and Their Cross-Reactivities
Pp. 835-845
K.Y. Jeong, C.-S. Hong and T.-S. Yong
[Abstract]
Polyproline Helices in Protein Structures: A Statistical
Survey Pp. 847-854
R. Berisio, S. Loguercio, A. De Simone, A. Zagari and
L. Vitagliano
[Abstract]
Crystallization Reports
Crystallization and Preliminary Crystallographic
Analysis of Allograft Inflammatory Factor 1 Pp. 855-858
S. Li, X. Liao, Z. Chen and M. Bartlam
[Abstract]
Expression, Purification, Crystallization, and
Preliminary X-Ray Analysis of the Human UDP-Glucose Dehydrogenase
Pp. 859-862
J.W. Huh, R.C. Robinson, H.S. Lee, J.I. Lee,
Y.S. Heo, H.T. Kim, H.J. Lee, S.W. Cho and H. Choe
[Abstract]
Abstracts
[Back to top]
Purification and Structural Characterization of Human
ERp29
J. Zheng, X. Liu, X. Yan, L. Dai and C. Ji
ERp29 is a major resident of the endoplasmic reticulum
(ER) and is postulated to play an important molecular chaperone
role in most animal cells. Human ERp29 was isolated to homogeneity
in high yield by using a bacterial expression system. Its
secondary structure was studied by circular dichroism (CD),
Fourier transformed infrared spectroscopy (FTIR) and Raman
spectroscopy and it was found that human ERp29 comprises significant
_-helical
structure. The details of its temperature-induced conformational
changes was studied by CD and FTIR for the first time, revealing
that the protein is stable below 50 °C and has two distinct
structural transitions between 50 °C and 70 °C. This
may shed light on ERp29’s inability to protect substrate
proteins against thermal aggregation.
[Back to top]
A Simple Search of TM Segments in Polytopic Membrane
Protein Using Matrix-Assisted Laser Desorption Ionization
Time-of-Flight Mass Spectrometry
Y. Abe, T. Hamasaki, S. Turusaki, S. Takazaki, J. Xiui,
D. Kang and N. Hamasaki
Using both high performance liquid chromatography (HPLC)
and amino acid sequencing (AAS), we previously analyzed band
3 TM peptide-segments that make up the transmembrane protein
structure. However, the HPLC/AAS combination method was highly
time-consuming. Matrix-Assisted Laser Desorption Ionization
Time-of-Flight (MALDI-TOF) mass spectrometry is used to obtain
accurate molecular weight information for proteins/peptides
simply and sensitively. We applied the MALDI-TOF mass spectrometry
technique to search for TM segments in membrane proteins.
In combination with trypsin cleavages after alkali treatments
(pH12 or 13) and sample preparation using organic solvents
for MALDI-TOF mass spectrometry, we determined the TM segments
of band 3 and glycophorin A in erythrocyte membrane. The method
can be applied to other polytopic membrane proteins in erythrocyte
membrane.
[Back to top]
The Stability Curve of Hen Egg White Lysozyme
S.S. Younvanich and B.M. Britt
The physiological stability curve -- a plot of the free
energy of unfolding versus temperature -- is calculated for
hen egg white lysozyme from a combination of extrapolated
unfolding thermodynamic data from reversible conditions and
isothermal titrations with guanidine hydrochloride. The shape
of the curve suggests the existence of only one folded conformation.
[Back to top]
Quantitative Analysis of Cytochrome C Released
from Rice Mitochondria Using the Adsorptive Polarographic
Wave of Guanidine Modified Co(II)-Cytochrome C Complex
C. Xia, X. Liu, D. Luo, H. Wang, F. He, L. Yuan and C.
Wang
A new, simple and sensitive method for the quantitative
analysis of cytochrome C (Cyt C) based on the reduction wave
of guanidine modified Co(II)-Cyt C complex at about –1.74
V (vs. SCE) by single sweep polarography in the solution containing
8×10-6
mol L-1 CoCl2, 0.04 mol L-1
guanidine hydrochloride, 0.2 mol L-1 NaOH and 0.5%
Na2SO3. The peak height is linearly
proportional to the concentration of Cyt C in the range of
0.005~1.500 mg L-1 (correlation coefficient 0.999).
Common amino acids, saccharide, organic acid and metal ions
of appropriate concentrations have no interference on the
Cyt C determination. The released Cyt C in the process of
mitochondrial permeability transition of Hong-Lian cytoplasmic
male sterile line of rice has been measured by the method,
and the result is satisfactory
[Back to top]
Isolation and Partial Characterization of Ribonuclease
Inhibitor from Goat Liver
J. Chatterjee, T.K. Maiti and S. Dasgupta
Ribonuclease inhibitor (RI), a 50 kDa protein, has been
found both in mammalian and nonmammalian tissues. We have
isolated RI from goat liver and partial characterization has
been accomplished. For the isolation of RI, DEAE cellulose
column chromatography followed by affinity chromatography
using CNBr activated Sepharose 4B was performed. The inhibition
of ribonucleolytic activity of Ribonuclease A has been checked
by an agarose gel based assay. The antiangiogenic property
of the protein was tested by the chorioallantoic membrane
(CAM) assay. Results indicate inhibition of angiogenesis.
[Back to top]
High-Level Expression and Purification of Human
Epidermal Growth Factor with SUMO Fusion in Escherichia
coli
Z. Su, Y. Huang, Q. Zhou, Z. Wu, X. Wu, Q. Zheng,
C. Ding and X. Li
Human epidermal growth factor (hEGF) can stimulate the
division of various cell types and has potential clinical
applications. However, the high expression of active hEGF
in Escherichia coli has not been successful, as the
protein contains three intra-molecular disulfide bonds that
are difficult to form correctly in the bacterial intracellular
environment. To solve this problem, we fused the hEGF gene
with a small ubiquitin-related modifier gene (SUMO) by synthesizing
an artificial SUMO-hEGF fusion gene that was highly expressed
in Origami (DE3) strain. The optimal expression level of the
soluble fusion protein, SUMO-hEGF, was up to 38.9% of the
total cellular protein. The fusion protein was purified by
Ni-NTA affinity chromatography and cleaved by a SUMO-specific
protease to obtain the native hEGF, which was further purified
by Ni-NTA affinity chromatography. The result of the reverse-phase
HPLC showed that the purity of the recombinant cleaved hEGF
was greater than 98%. The primary structure of the purified
hEGF was confirmed by N-terminal amino acid sequencing and
MALDI-TOF mass spectroscopy analysis. Using the method of
methylthiazoletetrazolium, the mitogenic activity on Balb/c
3T3 cells of the purified hEGF was comparable to that of commercial
hEGF.
[Back to top]
Effects of Physical, Ionic, and Structural
Factors on the Binding of Repressor of Mycobacteriophage L1
to Its Cognate Operator DNA
T. Ganguly, P.K. Chanda, A. Bandhu, P. Chattoraj,
M. Das and S. Sau
To determine the factors influencing the binding of L1
repressor to its cognate operator DNA, several gel shift as
well as bioinformatic analyses have been carried out. The
data show that time, temperature, salt, and pH each greatly
affect the binding. In order to achieve optimum operator binding
of L1 repressor in Tris buffer, the minimum requirements of
time, temperature, salt, and pH were estimated to be 1 min,
32°C, NaCl (50 mM), and 7.9, respectively. Interestingly
Na+ but not NH4+, K+,
or Li+ was found to augment significantly the binding
activity of CI protein above the basal level. Anions like
Cl-, citrate-, acetate-,
and H2PO4- do not alter the
binding of L1 repressor to its operator. We also show that
an in frame deletion mutant of L1 repressor which does not
carry the putative HTH motif (at its N-terminal end) fails
to bind to its cognate operator DNA even at very high concentrations.
The putative HTH motif was found highly conserved and evolutionarily
very close to that of regulatory proteins of Y. pestis,
H. marismortui, A. tumefaciens, etc. Taken together we
suggest that N-terminal end of L1 repressor carries a HTH
motif. Further analysis of the putative secondary structures
of mycobacteriophage repressors reveals that two common regions
encompassing more than 90% of primary sequence are present
in all the four repressor molecules studied here. The results
suggest that these common regions are utilized for carrying
out identical functions.
[Back to top]
Purification and Thermal Characterization of
a Novel Peroxidase from a Local Chick Pea Cultivar
H.N. Bhatti, A. Najma, M. Asgher, M.A. Hanif
and M.A. Zia
A novel peroxidase isolated from a local chick pea (Cicer
arietinum L.) cultivar (Balksar 2000) was purified by
means of ammonium sulfate precipitation, DEAE-cellulose chromatography
and two runs on gel filtration. The purified enzyme has a
specific activity of 2045 U/mg with 17 % activity recovery.
The molecular mass of the enzyme was estimated to be 39 kDa
by SDS-polyacrylamide gel electrophoresis. Optimum pH and
temperature of the enzyme were 5.5 and 45°C respectively.
The thermal denaturation of local chick pea peroxidase was
studied in aqueous solution at temperatures ranging from 45°C
to 65°C. The temperature of 50% inactivation of the enzyme
was found to be 68°C. The enthalpy (ΔH*) and free
energy (ΔG*) of thermal denaturation of chick pea peroxidase
were 101.4 and 103.4 k J/mol respectively at 65°C.Metals
like Zn2+, Mn2+, Hg2+, Co2+
and Al3+ slightly inhibited the peroxidase activity
while Ca2+, Mg2+ and Ba2+
have no effect on enzyme activity. The high specific activity
and thermal stability make chick pea peroxidase an alternative
to horseradish peroxidase (HRP) in various applications.
[Back to top]
Antimicrobial Properties of Nuclear Diffusion
Inhibitory Signal of HIV-1 Rev
N. Kobayashi and T. Yoshida
Nuclear Diffusion Inhibitory Signal (NIS) has been identified
in human immunodeficiency virus type 1 (HIV-1) Rev as a nuclear
signal peptide which modulates nucleocytoplasmic protein trafficking
and intracellular stability of the HIV-1 Rev. In this study,
it was discovered that antimicrobial properties are inherent
in the NIS. This is a significant finding that the NIS, which
does not exist solely for self defense, in fact possesses
antimicrobial properties.
[Back to top]
In Silico Criterion for Prediction of
Effects of p53 Gene Missense Mutations on p53-Mdm2 Feedback
Loop
N. Veljkovic and V. Perovic
The Informational Spectrum Method (ISM) is the tool for
the in silico analysis of proteins which interprets
protein sequence linear information using signal analyses
methods. In this paper the ISM was employed to characterize
the products of genetic variants of tumor suppressor gene
p53 and its natural binding regulator protein Mdm2. Based
on this we propose the criterion for identification of missense
mutations that have impact on the p53-Mdm2 feedback loop.
The efficiency of the proposed criterion was confirmed by
the ISM analyses of p53 mutants reported in: (i) healthy individuals,
(ii) germline mutations database and (iii) somatic mutations
database.
[Back to top]
N-Terminal Mutational Analysis of the Interaction
Between Growth-Blocking Peptide (GBP) and Receptor of Insect
Immune Cells
S. Watanabe, M. Tada, T. Aizawa, M. Yoshida, T. Sugaya,
M. Taguchi, T. Kouno, T. Nakamura, M. Mizuguchi, M. Demura,
Y. Hayakawa and K. Kawano
GBP, a small insect cytokine isolated from lepidopterans,
has a variety of functions. We constructed a series of mutants
focusing on the unstructured N-terminal residues of GBP by
acetylation, deletion, and elongation in order to investigate
the interaction between GBP and its receptor in plasmatocytes.
The 1H NMR spectra showed no significant changes
in the tertiary structures of these peptides, which indicated
that all the mutants maintained their core β–sheet
structures. The deletion and acetylated mutants, 2-25GBP,
Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the
strongest antagonist, while Ac2-25GBP and AcGBP were moderate.
In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and
(-2G,-1R)GBP maintained their plasmatocyte-spreading activity.
These results demonstrate the importance of the GBP N-terminal
charged amine and length of N-terminal GBP-peptide backbone
for plasmatocyte-spreading activity. Next, we analyzed other
mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on
Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading
activity, whereas 2-25GBP lost its activity. Finally, substituted
mutant, F3AGBP, had neither plasmatocyte-spreading activity
nor antagonistic activity. These results demonstrate the function
of each N-terminal residue in the interaction between GBP
and its receptor in plasmatocytes.
[Back to top]
Expression and Purification of Exendin-4
Dimer in Escherichia coli and Its Interaction with
GLP-1 Receptor In Vitro
L. Yi, X. Yin, D. Wei and Y. Ma
Exendin-4 is a 39 amino acid peptide isolated from the
Gila monster salivary gland. It is 53% homologous to GLP-1
and exhibits similar glucoregulatory activities. In this study,
exendin-4 dimer (D-Ex4) was constructed, cloned into plasmid
pET32a(+) and expressed in E. coli BL21(DE3). The
fusion protein with His-tag at the N-terminus was purified
with a Ni-NTA-agarose column. After proteolytic cleavage,
D-Ex4 peptide with high purity was obtained by HPLC. The results
obtained by chemical cross-linking showed that D-Ex4 maintained
affinity to GLP-1 receptor.
[Back to top]
Transformation of a Biologically Active Peptide
into Peptoid Analogs While Retaining Biological Activity
B. Hoffmann, T. Ast, T. Polakowski, U. Reineke
and R. Volkmer
We report the stepwise transformation of a linear peptide
epitope recognized by the anti-transforming growth factor
α monoclonal
antibody Tab2 into peptomers and finally into peptoid analogs.
The key experiment in this study is the substitution analysis
in which each position of the peptide is exchanged by a set
of different peptoid building blocks resulting in a peptidomimetic
array. After probing the array toward antibody binding, the
best binding peptomer spots were selected and subjected to
a successive transformation. The best peptoid found in this
study has a KD of 200 nM when binding to Tab2,
which is only 8-fold higher than the starting peptide. Moreover,
this approach permits to ask directly questions about the
transformation of peptide lead structures into non-peptidic
compounds in the context of protein recognition.
[Back to top]
Allergenic Tropomyosins and Their Cross-Reactivities
K.Y. Jeong, C.-S. Hong and T.-S. Yong
The ingestion or inhalation of some proteins may lead
to adverse immune reactions. Allergens may trigger allergic
reactions in genetically predisposed individuals when they
are absorbed through the skin or make contact with mucous
membranes. An allergic disease often deteriorates the quality
of life and may sometimes be life-threatening due to anaphylactic
shock.
A number of allergens have been characterized from various
multicellular organisms to date. It is thought to be reasonable
to pay a special attention to the substance which is highly
cross-reactive and which causes adverse responses in the molecules
that are not sensitized but similar to the sensitized allergen.
Tropomyosin has been described as an important food allergen
in shrimp, lobster, crab, oysters, squid, and other invertebrates.
Allergic reactions to shellfish and mollusks are often cross-reactive,
which may be explained by the highly conserved amino acid
sequences of tropomyosins among invertebrates, but vertebrate
tropomyosins are not known to be allergenic. Several tropomyosins
from domestic arthropods have been reported to be allergenic.
Recently, it was suggested that an infection of helminthic
parasites might lead to sensitization to tropomyosin and elicit
allergic reactions to other invertebrates.
Much effort has been made to characterize these allergenic
tropomyosins from various sources. We will discuss the physicochemical
characteristics and the potential application of tropomyosin
for the diagnosis and therapeutics of allergic disorders.
[Back to top]
Polyproline Helices in Protein Structures: A
Statistical Survey
R. Berisio, S. Loguercio, A. De Simone, A. Zagari and
L. Vitagliano
A statistical survey of polyproline II (PPII) helices
extracted from protein crystal structures is here reported.
The average hydrophobicity of these helices is intermediate
between those displayed by β-strands
and coil regions and is similar to that of α-helices.
PPII helices with amphipathic properties have been identified
and classified. Amino acid propensities for PPII helices derived
in this study differ significantly from those previously reported.
They show a little albeit significant correlation with propensities
for α-helices
whereas they are fully non-correlated to propensities for
β-sheets.
Finally, PPII propensities have been correlated with amino
acid frequencies in structural proteins, such as collagen
and extensins.
[Back to top]
Crystallization and Preliminary Crystallographic
Analysis of Allograft Inflammatory Factor 1
S. Li, X. Liao, Z. Chen and M. Bartlam
Allograft inflammatory factor-1 (AIF-1) is a 17-kDa IFN-γ
inducible Ca2+-binding EF-hand protein involved
in immune dysfunction and smooth muscle cell activation. AIF-1
was solubly expressed in E. coli and purified. Crystals
of AIF-1 were grown at 291 K using PEG-8000 as precipitant.
Diffraction by the AIF-1 crystal extends to 3.3 Å resolution,
and the crystal belongs to the space group P43
with unit cell parameters a=b=73.4, c=49.1 Å.
[Back to top]
Expression, Purification, Crystallization, and
Preliminary X-Ray Analysis of the Human UDP-Glucose Dehydrogenase
J.W. Huh, R.C. Robinson, H.S. Lee, J.I. Lee,
Y.S. Heo, H.T. Kim, H.J. Lee, S.W. Cho and H. Choe
UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis
of UDP-glucuronic acid from UDP-glucose resulting in the formation
of proteoglycans that are involved in promoting normal cellular
growth and migration. Overproduction of proteoglycans has
been implicated in the progression of certain epithelial cancers.
Here, human UGDH (hUGDH) was purified and crystallized from
a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate,
pH 6.5, and 21% PEG 8000. Diffraction data were collected
to a resolution of 2.8 Å. The crystal belongs to the
orthorhombic space group P212121
with unit-cell parameters a = 173.25, b = 191.16, c = 225.94
Å, and α
= β
= γ
= 90.0°. Based on preliminary analysis of the diffraction
data, we propose that the biological unit of hUGDH is a tetramer.
|
