Protein & Peptide Letters, Volume 11, No. 1, 2004
Spectroscopic
Characterization Of Phaseolus
Vulgaris Leucoagglutinin Pp.1-7
Shyamasri Biswas and Arvind M. Kayastha
The
Primary Ligand-Binding Interaction At
The Glp-1 Receptor Is Via The Putative Helix Of The Peptide Agonists Pp.9-14
Suleiman
Al-Sabah and Dan Donnelly
Met-204
And Tyr-205 Are Together Important For Binding Glp-1
Receptor Agonists But Not Their N-Terminally Truncated Analogues Pp.15-22
Rakel Lopez de Maturana,
Janet Treece-Birch, Fatima Abidi,
John B.C.
NMR Studies Of The Prionogenic Peptide Derived From Sup35 Protein Pp.23-28
Young
Kee Chae, Kyunghee Lee, Yongae Kim
Cytosolic Insulin-Binding Proteins Of Mouse
Liver Cells Pp.29-33
Petr G. Lokhov, Sergei A. Moshkovskii, Olga M. Ipatova, Vladimir N. Prozorovskii
Effects
Of Two Glycine Residues In
Positions 13 And 17 Of Pleurocidin On Structure And
Bacterial Cell Selectivity
Pp.35-40
Shin
Saeng Lim, Yun Mi Song, Mi Hyang Jang, Yangmee Kim, Kyung-Soo Hahm and Song Yub Shin
Quaternary
Structure Of a-Crystallin Is Necessary For The
Binding Of Unfolded Proteins: A Surface Plasmon
Resonance Study Pp.41-48
Expression
And Secretion Of Functional Recombinant 1 Scu-PA:AV In Insect Cell Using Signal Peptides Pp.49-55
Seed
Protein Variation Among Pepper (Capsicum Sp.)
Genotypes Revealed By Malditof Analysis Pp.57-62
Expression,
Purification And Characterization Of Recombinant Mouse
PC6B From Baculovirus-Infected Insect Cells Pp.63-69
Fluorescent
Modification Of N-Terminal Amino Group In Peptides For
Complete Sequence Analysis Using PSD Method In MALDI-TOF-MS Pp.71-77
Crystallization
And X-Ray Crystallographic Studies Of An Inhibitor From Rye (Secale Cereale) Active Against Acanthoscelides Obtectus And Zabrotes Subfasciatus
Alpha-Amylases Pp.79-83
Biophysical
Characterization Of An Insect Lysozyme
From Manduca Sexta Pp.85-92
Crystallization
And Mad Data Collection Of High-Molecular Weight
Cytochrome C From Desulfovibrio Vulgaris
Miyazaki F Pp.93-96
[Back to top] Spectroscopic
Characterization Of Phaseolus
Vulgaris Leucoagglutinin
Shyamasri Biswas and Arvind M. Kayastha
Phaseolus vulgaris leucoagglutinin is a homotetrameric
legume lectin possessing the canonical dimeric
structure common to other legume lectins. In order to
gain insight into the stability of the protein in an acidic environment, it was
characterized by CD and fluorescence studies at pH 2.5. This was then compared
with the native protein at physiological pH (7.2). The extinction coefficient
of the native protein was calculated to be 3.58x104 from its UV absorption
spectra. The far- and near-UV CD spectra of the protein at pH 2.5 showed very
little difference even though the protein was found to exist as a dimer at pH 2.5. The fluorescence emission maxima of the
protein upon excitation at 280 nm were found to shift only from 331 nm at pH
7.2 to 333 nm at pH 2.5. Based on the above observation it was concluded that
the protein exhibits extreme pH stability especially in the acidic range. The
secondary and tertiary structure of the protein is lost only when it is
incubated for two days in 6 M GdnHCl at pH 2.5. At pH
7.2 it could be denatured in 6 M GdnHCl after one
week of incubation.
[Back to top] The
Primary Ligand-Binding Interaction At
The Glp-1 Receptor Is Via The Putative Helix Of The Peptide Agonists
Suleiman
Al-Sabah and Dan Donnelly
The N-terminal
domain of the GLP-1 receptor binds the putative helical region of the peptide
agonists, GLP-1 and exendin-4. Here we demonstrate that this interaction also
determines the magnitude of a separate interaction between the N-terminus of these
peptides and the receptor’s core domain. Enhancing the pre-formation of the
C-terminal Trp-Cage motif of exendin-4, a motif
critical for high-affinity binding, results in no improvement in receptor
affinity, suggesting that this motif forms after the initial peptide-receptor
binding event.
[Back to top] Met-204 And
Tyr-205 Are Together Important For Binding Glp-1 Receptor Agonists But Not
Their N-Terminally Truncated Analogues
Rakel Lopez de Maturana, Janet Treece-Birch, Fatima Abidi, John B.C. Findlay and Dan Donnelly
A mutagenesis
study to systematically analyse residues spanning the
first extracellular loop of the GLP-1 receptor
identified a double mutant, Met-204/Tyr-205-Ala/Ala, which displayed: markedly
reduced affinity for the natural agonist GLP-1; slightly reduced affinity for
its analogue exendin-4; and unaltered affinity for several N-terminally
truncated analogues of GLP-1 and exendin-4. This suggests that the locus is
important for the formation of the binding site for the N-terminal residues of
peptide agonists.
[Back to top] NMR Studies Of The Prionogenic
Peptide Derived From Sup35 Protein
Young
Kee Chae, Kyunghee Lee, Yongae Kim
The NMR studies of
the prionogenic peptide derived from Sup35 are
presented. The peptide molecules were dissolved in the half-aqueous solution to
prevent severe aggregation, and were found to be in an extended conformation
from the chemical shift and the coupling constant data. They could form higher order
multimers by making intermolecular hydrogen bonds,
judging from the observation that the NMR sample became a gel-like state at
lower temperatures. This work reports the first structural information in the
solution state about the prionogenic peptide mimicking
the state of amyloid fibrils, and provides a solid
foundation for further structural analysis of peptide molecules forming
insoluble aggregates.
[Back to top] Cytosolic
Insulin-Binding Proteins Of Mouse Liver Cells
Petr G. Lokhov, Sergei A. Moshkovskii, Olga M. Ipatova, Vladimir N. Prozorovskii
It has been
recently shown that insulin retains its biological activity after
receptor-directed internalization and it may affect the cell metabolism by
interaction with cytosolic insulin-binding proteins (CIBPs). Using affinity chromatography combined with
SDS-PAGE and MALDI-TOF mass-spectrometry we have identified 7 proteins from
mouse liver cells that specifically bind to the insulin, including adenylate kinase 2 (25.6 kD), kinesin superfamily
protein 20B (26.0 kD), hepatic arginase
1 (34.8 kD), fructose-bisphosphate
aldolase B (39.5 kD),
4-hydroxyphenylpyruvate dioxygenase (45.1 kD), betaine-homocysteine methyl-transferase (45.0 kD) and KRIT1
(83.4 kD).
[Back to top] Effects Of
Two Glycine Residues In Positions 13 And 17 Of Pleurocidin On Structure And Bacterial Cell Selectivity
Shin
Saeng Lim, Yun Mi Song, Mi Hyang Jang, Yangmee Kim, Kyung-Soo Hahm and Song Yub Shin
Pleurocidin (Ple), a 25-residue
a-helical antimicrobial peptide, isolated from skin mucosa of the winter
flounder, shows potent bacterial cell selectivity. In this study, the effect of
two glycine residues in positions 13 and 17 of Ple on structure and bacterial cell selectivity was
investigated by Gly®Ala substitution. Ala-substitution (Gly13, 17®Ala,
Gly13 ®Ala and
Gly17® Ala) in positions 13 and 17 of Ple did not induce a significant change in antibacterial
activity, but increased hemolytic activity. Both Gly13 ®Ala and
Gly17 ®Ala substitution did not cause a remarkable
change in a-helical content in SDS micelles, while Gly13,17®Ala
substitution caused a drastic increase in a-helical content. These results suggest that
the hinge region from Gly13 to Gly17 of Ple is
assumed to provide its conformational flexibility and bacterial cell
selectivity.
[Back to top] Quaternary Structure Of a-Crystallin Is Necessary For The
Binding Of Unfolded Proteins: A Surface Plasmon
Resonance Study
Sergiy V. Avilov, Nataliya A. Aleksandrova,
Alexander P. Demchenko
The interactions
between an oligomeric heat-shock protein, a-crystallin, and
its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed
us for the first time to study isolated a-crystallin
subunits under physiological conditions. We observe that these subunits, in
contrast to a-crystallin oligomers,
do not bind unfolded protein. Our data indicate that quaternary structure of a-crystallin is
necessary for its chaperone-like activity.
[Back to top] Expression And Secretion Of
Functional Recombinant 1 Scu-Pa:Av
In Insect Cell Using Signal Peptides
Yuhui Huang, Ruiyang Tian, Wei Hu,
Xiangfu Wu, Shengli Yang and Yi Gong
A fusion protein (scu-PA:AV) was expressed in the baculovirus expression system and secreted from Sf9 cells
lead by signal peptides, 2mel and 3egt. The scu-PA:AV displays both the urokinase
activity and membrane binding activity of its parental components. Our work
indicated that it is possible to be developed as a thrombus-targeting drug.
[Back to top] Seed Protein Variation Among Pepper (Capsicum Sp.) Genotypes Revealed By Malditof Analysis
Marco
Aurelio Caldas de Pinho Pessoa Filho, Carlos Bloch
Junior, Danilo Fernandes da Silva Filho, Alexsandro Sobreira Galdino, Rodrigo Maranguape Silva
da Cunha, Maria Aparecida Oliveira Alves and Thalles Barbosa Grangeiro
A method for seed
proteome analysis using MALDI-TOF mass spectrometry is described. The data were
used to estimate the genetic diversity degree among twelve genotypes of pepper
(Capsicum). The resulting spectra were converted into a binary matrix
consisting of 23 protein data sets, and genetic similarity values were
calculated with the FreeTree software and Jaccard’s coefficient of similarity. We have also been able
to identify the presence of certain proteins in the extracts, by checking their
masses on on-line databases.
[Back to top] Expression, Purification And Characterization Of Recombinant Mouse PC6B From Baculovirus-Infected Insect Cells
Lie
Wang, Guanzhen Yang, Xiangfu
Wu
we constructed a mouse PC6B truncated mutant
and introduced a tag of 6 consecutive histidines at
its carboxyl terminus for simple purification. Using the baculovirus
expression system and standard enzymatic assay, we
obtained recombinant mouse PC6B protein and with enzymatic activity.
[Back to top] Fluorescent Modification Of N-Terminal Amino Group In Peptides For Complete Sequence
Analysis Using PSD Method In MALDI-TOF-MS
Masatoshi
Nakagawa and Hiroshi Nakanishi
In the sequence
analyses of peptides from nucleoprotein in influenza virus, it was very
difficult to obtain the sufficient numbers of fragment ions using the
post-source decay (PSD) method in MALDI-TOF-MS. Fluorescent modification of the
N-terminal amino group in the target peptides was introduced for the PSD
measurement. The fluorescently-labelled peptides gave
sufficient fragment ions in the PSD spectrum, which leads to the complete
sequence analysis of peptide.
[Back to top] Crystallization And X-Ray Crystallographic Studies Of An Inhibitor From Rye
(Secale Cereale) Active
Against Acanthoscelides Obtectus
And Zabrotes Subfasciatus
Alpha-Amylases
Jorge
Iulek, Christiane Trevisan Slivinski and Marcio Silva
Crystals of a new
inhibitor present in rye seeds active against alpha-amylases from crop pests Acanthoscelides obtectus and Zabrotes subfasciatus have been
obtained. A native dataset was collected at 2.21 Å resolution
with 99.3 % completeness at CPr beamline
at LNLS. The crystals belong to the trigonal system,
space group P3121 with a = b = 78.21 Å, and c = 59.61 Å. The crystal calculated
solvent content is compatible with one dimer per
asymmetric unit.
[Back to top] Biophysical Characterization Of An Insect Lysozyme From Manduca Sexta
Alonso
A. Lopez-Zavala, Enrique de-la-Re-Vega, Sergio A. Calderon-Arredondo, Karina D. Garcia-Orozco, Enrique F. Velazquez, Maria A. Islas-Osuna, Miguel A. Valdez and Rogerio
R. Sotelo-Mundo
Insect lysozyme from Manduca sexta (MS-lys) was overexpressed in E. coli and refolded to obtain active
protein. Recombinant MS-lys presented a globular
structure, with an alpha-helical content of 57% as assessed by circular dichroism spectroscopy. Light scattering studies showed
that in solution MS-lys has a quasi-monodisperse size distribution, with a rod-like structure
similar to nucleation clusters reported in egg lysozyme
pre-crystallization stages. These results show that MS-lys
is an excellent candidate for crystallization, folding and denaturation
studies.
[Back to top] Crystallization And Mad Data Collection Of High-Molecular Weight Cytochrome
C From Desulfovibrio Vulgaris
Miyazaki F
Naoki
Shibata, Kyoko Suto, Eiko Ichimura, Kazutaka Yoshimura,
Kenji Muneo, Shoko Tomigami,
Yukio Morimoto, Mari Ogata, Tatsuhiko Yagi, Yoshiki Higuchi and Noritake Yasuoka
Hexadecaheme high molecular weight cytochrome
c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and
crystallized. X-ray diffraction data have been collected by the multiple
wavelength anomalous dispersion method. The crystal belongs to the space group
P212121 with unit-cell parameters a = 60.42, b = 84.29 and c = 144.16 Å and
contains one molecule per asymmetric unit.