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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 13, Number 1, 2006
Contents
Regular Papers
Membrane Lipid Composition and the Interaction
of Pardaxin: The Role of Cholesterol Pp. 1-5
R.F. Epand, A. Ramamoorthy and R.M. Epand
[Abstract]
A Three-Dimensional Homology Model of
the O-Acetylserine Sulfhydrylase-B from Salmonella
typhimurium Pp. 7-13
W.M. Rabeh, T. Mather and P.F. Cook
[Abstract]
Novel Plastocyanin Containing Phenyl-alanine-83 from
the Gymnosperm Ginkgo biloba Pp.
15-19
J. Onodera, Y. Sugimura and F. Yoshizaki
[Abstract]
The Cardioprotective Peptide, Urocortin, Activates
Cardiac KATP Channels in Adult Rat. Pp.
21-27
H. Xu, Y. Wang, J. Tao, C. Yang, J. Chen, H. Wang and S. Li
[Abstract]
Fragment Library-Based Representation System for
Protein Conformation Pp. 29-31
R. Kim and C.-Y. Choi
[Abstract]
Sequential Events in Ribonuclease A Thermal Unfolding
Characterized by Two-Dimensional Infrared Correlation Spectro-scopy
Pp. 33-40
J. Zhang, H.-W. He, Q Wang and Y.-B. Yan
[Abstract]
Expression and Purification of a Putative Tumor
Suppressor Gene PP5715 in E. coli with Growth
Inhibition of Hepatocellular Carcinoma Cells Pp.
41-45
M. Wen-Zhe, H. Wei, Q. Wen-Xin, W. Da-Fang, G. Jian-Ren, Y.
Sheng-Li and G. Yi
[Abstract]
Structure Study of Recombinant RGD-Hirudin by
Vibrational and Circular Dichroism Spectroscopy
Pp. 47-51
X. Liu, W. Mo, L. Dai, X. Yan and H. Song
[Abstract]
Antibiotic Activity of Antimicrobial Peptide Against
Pseudomonads Isolated from a Patient with Gallstones
Pp. 53-58
Y. Park, J. Soo Hahm and K.-S. Hahm
[Abstract]
Predominant Expression of rTERTb, an Inactive
TERT Variant, in the Adult Rat Brain Pp.
59-65
R. Kaneko, S. Esumi, T. Yagi and T. Hirabayashi
[Abstract]
Oxidative Folding of Conotoxins in the Immobilized
Systems Pp. 67-70
B.R. Green and G. Bulaj
[Abstract]
Biophysical Characterization of the Non-Fusogenic
Interaction Between Liposomes and the Shrimp Bifunctional
Lipoprotein-β-Glucan Binding Protein HDL/BGBP Pp.
71-75
E.F. Morán-Palacio, M.A. Tricerri, K. García-Orozco,
M.G. Romo-Figueroa, G. Yepiz-Plascencia, H.A. Garda and R.R.
Sotelo-Mundo
[Abstract]
The Monomers of the P2X1 Receptor Model and KcsA
Protein Share a Similar Structural Fold. Pp. 77-81
P.P. Mager, A. Weber, L. Sanchez, K. Wirkner and P. Illes
[Abstract]
Preliminary Functional Characterization, Cloning
and Primary Sequence of Fastuosain, a Cysteine Peptidase Isolated
from Fruits of Bromelia fastuosa Pp.
83-89
H. Cabral, A.M. Leopoldino, E.H. Tajara, L.J. Greene, V.M.
Faça, R.P. Mateus, C.R. Ceron, W.A. de Souza Judice,
L. Juliano and G.O. Bonilla-Rodriguez
[Abstract]
Ca2+ Binding Effects on the
C2 Domain Conformation of Human Cytosolic Phospho-lipase
A2 Pp. 91-94
B. Tan, S.-B. Qin, M.-E. Chen, H.-X. Cang and H.-J. Zhang
[Abstract]
Potential for Ultrafast Protein Separations with Capillary-Channeled
Polymer (C-CP) Fiber Columns Pp. 95-99
D.M. Nelson and R.K. Marcus
[Abstract]
Pattern of Positions Sensitive to Mutations in
Human Haemoglobin α-Chain
Pp. 101-107
N. Gao, S. Yan and G. Wu
[Abstract]
Abstracts
[Back to top]
Membrane Lipid Composition and the Interaction of
Pardaxin: The Role of Cholesterol
R.F. Epand, A. Ramamoorthy and R.M. Epand
Modulation by pardaxin of the phase transitions of dimyristoyl
phosphatidylcholine, 1-stearoyl-2-oleoyl phosphatidylcholine
or 1-stearoyl-2-oleoyl phosphatidylglycerol in the presence
or absence of cholesterol was studied by differential scanning
calorimetry. The transition enthalpy of each of the phospholipids
was lowered by pardaxin and there was a small decrease in
the transition temperature. Addition of cholesterol and pardaxin
to dimyristoyl phosphatidylcholine resulted in a very marked
lowering of the transition temperature. Although the peptide
broadens the transition of the pure phospholipids, it sharpens
the transition of mixtures of the phospholipids with cholesterol.
This and the observation that pardaxin also causes the formation
of crystallites of anhydrous cholesterol, suggest that the
peptide promotes redistribution of cholesterol in the membrane.
[Back to top]
A Three-Dimensional Homology Model of the O-Acetylserine
Sulfhydrylase-B from Salmonella typhimurium
W.M. Rabeh, T. Mather and P.F. Cook
O-acetylserine sulfhydrylase (OASS) catalyzes the
last step in the cysteine biosynthetic pathway in enteric
bacteria and plants. The overall pathway involves the substitution
of the β-acetoxy
group of O-acetyl-L-serine with inorganic bisulfide.
Two isozymes are present in S. typhimurium, the A-
and B-isozymes, expressed under aerobic and anaerobic conditions,
respectively. No crystal structure is presently available
for the B-isozyme. Kinetic data indicate the catalytic mechanism
of OASS-B is ping-pong, as found for the A-isozyme, but kinetic
parameters and substrate specificity differ. In order to estimate
whether structural differences may be responsible for the
kinetic differences, a homology model was built using the
structure of OASS-A as the template for the OASS-B model.
The β-subunit
of tryptophan synthase and cystathionine β-synthase
were used for comparison. Differences between the OASS-A structure
and the homology model for OASS-B are discussed.
[Back to top]
Novel Plastocyanin Containing Phenyl-alanine-83 from
the Gymnosperm Ginkgo biloba
J. Onodera, Y. Sugimura and F. Yoshizaki
Plastocyanin was purified from the gymnosperm Ginkgo
biloba L., and its complete amino acid sequence was determined.
The protein was shown to contain Phe-83 instead of Tyr-83
conserved in other land plant plastocyanins. This is the first
report of the characterization and complete amino acid sequence
of a gymnosperm plastocyanin.
[Back to top]
The Cardioprotective Peptide, Urocortin, Activates
Cardiac KATP Channels in Adult Rat.
H. Xu, Y. Wang, J. Tao, C. Yang, J. Chen, H. Wang and S. Li
Urocortin (UCN), an endogenous cardioprotective peptide,
has been reported to activate several ion channels but its
effects on the cardiac KATP channels are unknown. In this
paper, we showed that adult rat cardiac myocytes had small
KATP currents that could be increased 20-fold by pinacidil.
UCN also concentration-dependently activated the KATP current
and this effect was influenced by ATP and PKA inhibitor peptide,
Rp-cAMPS.
[Back to top]
Fragment Library-Based Representation System for Protein
Conformation
R. Kim and C.-Y. Choi
The representation system for protein conformation has a
crucial effect on the speed of various protein-related simulations,
including ab initio protein structure prediction
and protein-protein docking simulation. Usually, the finer
a representation system, the longer is the computational time
required to employ the representation system in simulations.
On the other hand, very coarse lattice systems cannot be directly
applied to the simulation problems with real proteins. We
report a new, fragment library-based protein conformation
representation system, prepared by clustering amino acid conformations
from 154 proteins. This system was composed of 64 most representative
fragments per each amino acid, and based on the unified residue
approach in which two spheres per amino acid were used. It
could represent the conformation of the 82 proteins in an
independent test set with the mean and standard deviation
RMSD of 1.01 and 0.09 Å, respectively, based on the
position of alpha carbons and the centers of mass of sidechains.
[Back to top]
Sequential Events in Ribonuclease A Thermal Unfolding
Characterized by Two-Dimensional Infrared Correlation Spectro-scopy
J. Zhang, H.-W. He, Q Wang and Y.-B. Yan
The conformational changes in the thermal denaturation of
bovine pancreatic ribonuclease A was followed with infrared
spectra and analyzed by second derivative and two-dimensional
correlation techniques. By analyzing the sequential events
in each transition stage, the results were consistent with
a step-wise thermal denaturation mechanism in which the structural
adjustment of the N-terminal and the opening of the central
structure of the protein come before the main unfolding process.
Non-native turns were found to form along with the unfolding
of the native structures. The central region that is composed
of some β-sheet
and α-helical
structures was found to be the most stable part that might
form the residual structure at high temperatures.
[Back to top]
Expression and Purification of a Putative Tumor Suppressor
Gene PP5715 in E. coli with Growth Inhibition
of Hepatocellular Carcinoma Cells
M. Wen-Zhe, H. Wei, Q. Wen-Xin, W. Da-Fang, G. Jian-Ren, Y.
Sheng-Li and G. Yi
PP5715 is a putative tumor suppressor gene that
encodes a protein of 199 amino acids. Expression of PP5715
in E. coli was mainly as inclusion bodies. The recombinant
protein was purified by a NTA-Ni2+ affinity column,
refolded by dialysis, and shown to suppress growth of two
human hepatocellular carcinoma cell lines using the MTT assay
or the clonogenic assays. Computer analysis and fusion expression
of PP5175 and GFP showed that PP5715 may be a secretory protein.
It may bind to receptors on the membrane surface and thus
induce regulation of cell growth. These preliminary results
suggest that protein PP5715 may be a new tumor suppressor
with growth inhibition effects on hepatocellular carcinoma
cells.
[Back to top]
Structure Study of Recombinant RGD-Hirudin by Vibrational
and Circular Dichroism Spectroscopy
X. Liu, W. Mo, L. Dai, X. Yan and H. Song
The secondary structure of a new type of recombinant RGD-hirudin,
which has the activities of anti-thrombin and anti-platelet
aggregation, has been studied by Fourier transform infrared
spectroscopy (FT-IR), Raman spectroscopy and circular dichroism
(CD) methods. The distribution of various secondary structure
elements was determined using only a very small amount of
sample protein. It was found that the recombinant RGD-hirudin
contains about 26% extended chain, 21% β-turn
and 53% unordered structure, leaving no α-helix.
The results showed that the regular secondary structure of
recombinant RGD-hirudin is increased compared with wild-type
hirudin. The RGD segment that is located at the end of a long
arm of a β-sheet
is thought to play an important role in the additional function
of anti-platelet aggregation. Throughout the experiments,
FT-IR, Raman spectroscopy and CD generated mutually reinforcing
results.
[Back to top]
Antibiotic Activity of Antimicrobial Peptide Against
Pseudomonads Isolated from a Patient with Gallstones
Y. Park, J. Soo Hahm and K.-S. Hahm
We investigated the in vitro antibiotic activity
of the 19-amino acid antimicrobial peptide HP (2-20), derived
from the N-terminus of Helicobacter pylori Ribosomal
Protein L1 (RPL1), against antibiotic susceptible and resistant
pathogens from a patient with gallstones. HP (2-20) was active
against antibiotic-susceptible and antibiotic-resistant clinical
isolates of pathogens from a patient with gallstones, but
this peptide showed no hemolytic activity against normal human
erythrocytes. HP (2-20) acted synergistically with ciprofloxacin
against pathogenic bacteria. Fluorescence activated flow cytometry
revealed that the effect of HP (2-20) was dependent on energy
and salt concentration. In addition, scanning electron microscopy
showed that HP (2-20) caused significant morphological alterations
to the cell surface of pathogens. Using 16S rDNA sequences,
we found that isolates from bile were 100% homologous to Pseudomonas
aeruginosa. These findings suggest that HP (2-20) may be useful
clinically as an antibiotic against acquired pathogens from
patients with gallstones and against pathogens resistant to
other antibiotics.
[Back to top]
Predominant Expression of rTERTb, an Inactive TERT
Variant, in the Adult Rat Brain
R. Kaneko, S. Esumi, T. Yagi and T. Hirabayashi
The full-length and four alternatively spliced variant mRNA
transcripts of rat TERT were identified. One of the variant,
rTERTb, was predominantly expressed during brain development
in accordance with the decrease in telomerase activity. The
rTERTb mRNA encoded an inactive truncated protein. Alternative
splicing of TERT may be important during brain development.
[Back to top]
Oxidative Folding of Conotoxins in the Immobilized
Systems
B.R. Green and G. Bulaj
We tested two alternative oxidation strategies to produce
conotoxins α-GI
and μ-PIIIA.
The peptides were either reversibly immobilized on a solid
support and then oxidized, or the immobilized disulfide reagent
(CLEAR-OXTM) was used to oxidize the peptides.
Both strategies appeared more efficient at higher peptide
concentrations, consistent with pseudo-dilution effects.
[Back to top]
Biophysical Characterization of the Non-Fusogenic
Interaction Between Liposomes and the Shrimp Bifunctional
Lipoprotein-β-Glucan Binding Protein HDL/BGBP
E.F. Morán-Palacio, M.A. Tricerri, K. García-Orozco,
M.G. Romo-Figueroa, G. Yepiz-Plascencia, H.A. Garda and R.R.
Sotelo-Mundo
Shrimp High Density Lipoprotein-β-Glucan
Binding Protein (HDL/BGBP) has been studied by its role in
nutrition and innate defense. Although the mechanisms of lipid
loading are still unknown, HDL-BGBP binds and aggregates phospholipids
vesicles in vitro. To gain insights into the HDL-BGBP
mechanism of interaction with membranes, we have used fluorescence
spectroscopy and electron microscopy. Data show that HDL-BGBP
does not induce membrane fusion, leakage nor lipid exchange,
although microstructural changes are clearly observed. This
work supports a model where protein aggregation leads to liposome
clustering. Such interaction may be a critical factor for
the activation of the shrimp blood cell in vivo.
[Back to top]
The Monomers of the P2X1 Receptor Model and KcsA
Protein Share a Similar Structural Fold.
P.P. Mager, A. Weber, L. Sanchez, K. Wirkner and P.
Illes
There is evidence that the P2X1 receptor subunit is involved
in apoptosis, platelet aggregation, and smooth muscle contraction.
The conformation of the membrane-embedded, ligand-gated mouse
P2X1 glycoprotein, a monovalent-bivalent cation channel-forming
receptor, is predicted. The first step is based on secondary
structure prediction. The secondary structure is converted
into a three-dimensional geometry. Then, the secondary and
tertiary structures are optimized by using the quantum chemistry
RHF/3-21G minimal basic set and the all-atom molecular mechanics
AMBER96 force field. The fold of the membrane-embedded protein
is simulated by a suitable dielectric. The structure is refined
using a conjugate gradient minimizer (Fletcher-Reeves modification
of the Polak-Ribiere method). Although the mouse P2X1 receptor
subunit is more complex (388 amino acids) than the KcsA protein
(160 amino acids), the overall folds are similar. The geometry
optimized P2X1 receptor subunit is freely available for academic
researchers on e-mail request (PDB format).
[Back to top]
Preliminary Functional Characterization, Cloning and
Primary Sequence of Fastuosain, a Cysteine Peptidase Isolated
from Fruits of Bromelia fastuosa
H. Cabral, A.M. Leopoldino, E.H. Tajara, L.J. Greene, V.M.
Faça, R.P. Mateus, C.R. Ceron, W.A. de Souza Judice,
L. Juliano and G.O. Bonilla-Rodriguez
The present work reports the characterization of Fastuosain,
a novel cysteine protease of 25kDa, purified from the unripe
fruits of Bromelia fastuosa, a wild South American
Bromeliaceae. Proteolytic activity, measured using casein
and synthetic substrates, was dependent on the presence of
thiol reagents, having maximum activity at pH 7.0. The present
work reports cDNA cloning of Fastuosain; cDNA was amplified
by PCR using specific primers. The product was 1096pb long.
Mature fastuosain has 217 residues, and with the proregion
has a total length of 324 residues. Its primary sequence showed
high homology with ananain(74%), stem bromelain (66%) and
papain (44%).
[Back to top]
Ca2+ Binding Effects on the C2 Domain
Conformation of Human Cytosolic Phospho-lipase A2
B. Tan, S.-B. Qin, M.-E. Chen, H.-X. Cang and H.-J.
Zhang
It has been reported that the cooperative binding of calcium
ions indicated a local conformational change of the human
cytosolic phospholipase A2 (cPLA2) C2
domain (Nalefski et al., (1997) Biochemistry
36, 12011-12018). However its structural evidence is
less known (Malmberg et al., (2003) Biochemistry
42, 13227-13240). In this letter, life-time decay and
fluorescence quenching techniques were employed to compare
the calcium-induced conformational changes. The life-time
decay parameters and fluorescence quenching constant changes
were small between the apo- and holo-C2 domains when tryptophan
residue was excited at 295 nm. In contrast, the quenching
constant change was large, from 0.52 M-1 for the
apo-C2 to 8.8 M-1 for the holo-C2 domain, when
tyrosine residues were excited at 284 nm. Our results provide
new information on amino acid side chain orientation change
at calcium binding loop 3, which is necessary for Ca2+
binding regulated membrane targeting of human cytosolic phospholipase
A2.
[Back to top]
Potential for Ultrafast Protein Separations with Capillary-Channeled
Polymer (C-CP) Fiber Columns
D.M. Nelson and R.K. Marcus
Capillary-channeled polymer (C-CP) fibers are demonstrated
as a potential stationary phase for liquid chromatography
separation of protein mixtures. Separation of a synthetic
mixture of four proteins is accomplished within a 45-second
window using a conventional revered-phase (RP) gradient, at
a mobile phase flow rate of 7 mL/min (10,200 mm/min).
[Back to top]
Pattern of Positions Sensitive to Mutations in Human
Haemoglobin α-Chain
N. Gao, S. Yan and G. Wu
In this study, we used the distribution rank, which has
been developed by us over the past several years, to quantify
134 mutations in the human hemoglobin α-chain
in order to gain an insight into the general pattern in mutations.
The results suggest that the pattern presented by distribution
rank can approximately estimate the positions that are sensitive
to mutations in human hemoglobin α-chain.
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