| Protein
& Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 15, Number 4, 2008
Contents
Regular Papers
Purification and Characterization of Peroxidase from
Cauliflower (Brassica oleracea L. var. botrytis)
Buds Pp. 320-326
E. Köksal and I. Gülçin
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Expression of a Canavalia brasiliensis
Lectin (ConBr) Precursor in Pichia pastoris
Pp. 327-332
C.P.S. Carvalho, C.S. Rocha, D.R. Nepomuceno, J.T.A. Oliveira
and T.B. Grangeiro
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Article]
A Proteomic Approach to Study Escherichia
coli. Acetyl Esterase Interactors Unveil a Sequence Motif
Involved in Protein-Protein Interaction Pp. 333-340
C.D’Ambrosio, L. Mandrich, M. Rossi, A. Scaloni
and G. Manco
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Building Quantitative Relationship Between Changed
Sequence and Changed Oxygen Affinity in Human Hemoglobin β-Chain
Pp. 341-345
G. Wu and S. Yan
[Abstract] [Purchase
Article]
Characterization of Fluoroalcohols-Induced Intermediates
of Mucor miehei Lipase at Low pH Pp. 346-352
S. Fatima, A. Mishra, P. Sen and R.H. Khan
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Synthesis of Two Peptide Mimetics as Markers for
Chemical Changes of Wool’s Keratin During Skin Unhairing
Process Pp. 353-355
D. Danalev, M. Koleva, D. Ivanova, L. Vezenkov
and N. Vassilev
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Synthesis and Biological Evaluation of N-Phosphoryl
Dipeptide Derivatives As Potent Apoptosis Inducers
Pp. 356-359
X. Yu, C. Tan, N. Zhang, W. Xu and Y. Jiang
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The Interaction Between Cholesterol and Human
Serum Albumin Pp. 360-364
L. Peng, H. Minbo, C. Fang, L. Xi and Z. Chaocan
[Abstract] [Purchase
Article]
Equilibrium Unfolding Mechanism of Chicken Muscle
Triose Phosphate Isomerase Pp. 365-370
Y. Shi, J.-h. Liu, H.-j. Zhang and Y. Ding
[Abstract] [Purchase
Article]
Effect of Cosolvents on the Stabilization of Bioactive
Peptides from Bovine Milk α-Casein
Pp. 371-376
S. Srinivas and V. Prakash
[Abstract] [Purchase
Article]
Purification and Characterization of a Novel Peroxidase
from Bitter Gourd (Momordica charantia)
Pp. 377-384
A. Fatima and Q. Husain
[Abstract] [Purchase
Article]
Advanced Glycation: Implications in Tissue Damage
and Disease Pp. 385-391
A. Gasser and J.M. Forbes
[Abstract] [Purchase
Article]
Using the Concept of Chou’s Pseudo Amino
Acid Composition to Predict Apoptosis Proteins Subcellular
Location: An Approach by Approximate Entropy Pp.
392-396
X. Jiang, R. Wei, T. Zhang and Q. Gu
[Abstract] [Purchase
Article]
Identification of a Short Basic Peptide Motif
Able to Drive Copy Number Dependent Nuclear Accumulation of
a Linked Protein Pp. 397-401
C. Christophe-Hobertus and D. Christophe
[Abstract] [Purchase
Article]
Production, Purification and Characterization
of β-1,4
Endoglucanase from a Novel Bacterial Strain CTP-09 of a Bacillus
sp. Pp. 402-410
M. Saleem, M.S. Akhtar, R. Yasmin, M. Zahid, N.N. Malik,
M. Afzal and M.I. Rajoka
[Abstract] [Purchase
Article]
Crystallization Reports
Crystallization and Preliminary X-Ray Diffraction Analysis
of a Novel Mannose-Binding Lectin with Antiretroviral Properties
from Polygonatum cyrtonema Hua Pp. 411-414
J.-J. Ding, J.-k. Bao, D.-Y. Zhu, Y. Zhang and
D.-C. Wang
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Article]
Crystallization and Preliminary X-Ray Diffraction Studies
of a Psychrophilic Iron Superoxide Dismutase from Pseudoalteromonas
haloplanktis Pp. 415-418
A. Merlino, I.R. Krauss, I. Castellano, E. De Vendittis,
A. Vergara and F. Sica
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Crystallization and Preliminary X-Ray Crystallographic
Analysis of Galectin LEC-1 from Caenorhabditis elegans
Pp. 419-422
T. Itagaki, S. Nishizaki, K. Sekihashi, H. Kobayashi,
S.-i. Kidokoro, Y. Kezuka, Y. Arata, J. Hirabayashi, K.-i.
Kasai and T. Nonaka
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Purification, Crystallization and Preliminary
Crystallographic Analysis of CYP 195A2, a P450 Enzyme from
Rhodopseudomonas palustris Pp.
423-426
D. Guo, F. Xu, S.G. Bell, X. Pang, M. Bartlam and
L.-L. Wong
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Abstracts
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Purification and Characterization of Peroxidase from
Cauliflower (Brassica oleracea L. var. botrytis)
Buds
E. Köksal and I. Gülçin
Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase)
are part of a large group of enzymes. In this study, peroxidase,
a primer antioxidant enzyme, was purified with 19.3 fold and
0.2% efficiency from cauliflower (Brassica oleracea L.)
by ammonium sulphate precipitation, dialysis, CM-Sephadex
ion-exchange chromatography and Sephadex G 25 purification
steps. The substrate specificity of peroxidase was investigated
using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid)
(ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol),
1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol.
Also, optimum pH, optimum temperature, optimum ionic strength,
stable pH, stable temperature, thermal inactivation conditions
were determined for guaiacol/H2O2,
pyrogallol/H2O2,
ABTS/H2O2,
catechol/H2O2
and 4 methyl catechol/H2O2
substrate patterns. The molecular weight (Mw)
of this enzyme was found to be 44 kDa by gel filtration chromatography
method. Native polyacrylamide gel electrophoresis (PAGE) was
performed for isoenzyme determination and a single band was
observed. Km and Vmax
values were calculated from Lineweaver-Burk graph for each
substrate patterns.
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Expression of a Canavalia brasiliensis
Lectin (ConBr) Precursor in Pichia pastoris
C.P.S. Carvalho, C.S. Rocha, D.R. Nepomuceno,
J.T.A. Oliveira and T.B. Grangeiro
A precursor of ConBr, a glucose/mannose-binding plant
lectin, was expressed in the yeast Pichia pastoris.
Western blot analysis of transformed cells detected an intracellularly
recombinant protein band with ca. 34.5 kDa. The recombinant
protein was apparently active as suggested by its strong interaction
with the mannose-rich yeast cell debris.
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A Proteomic Approach to Study Escherichia coli.
Acetyl Esterase Interactors Unveil a Sequence Motif Involved
in Protein-Protein Interaction
C.D’Ambrosio, L. Mandrich, M. Rossi, A. Scaloni
and G. Manco
E. coli acetyl esterase (Aes) and β-cysthationase
(MalY) interact, probably with the same mechanism, with the
N-terminus of transcriptional activator of maltose regulon
(MalT). In order to investigate the basic mechanism of this
interaction, we used both a proteomic and a bioinformatic
approach. Affinity-based mass spectrometry experiments with
purified Aes protein as bait allowed to fish twenty-three,
apparently specific, interactors from crude extracts of E.
coli cells grown in different conditions. The group of
interactors appeared quite heterogeneous, comprising Aes itself,
some molecular chaperons, metabolic enzymes, and several proteins
of unknown function. Among the identified proteins, two are
in some way related to the maltose metabolism and two are
related to the lipopolysaccharide metabolism. By superposing
the structures of the Alicyclobacillus acidocaldarius
EST2, an Aes homolog, and MalY, a region of structural similarity
was discovered that allowed detecting a short stretch of nine
residues with sequence similarity among EST2, AES and MalY.
Degenerated sequence consensuses derived from the alignment
were used to analyse the E. coli proteome in the
Swiss Prot database and permitted to retrieve sequences of
Aes interactors already known or detected in this study. Most
of these interactors (14 out of 25) contain the expected consensus.
A site-directed mutant of Aes R179A made in the consensus
sequence resulted in complete loss of interaction. Based on
the analysis of the available three-dimensional structures
and mutagenic and structural data inferred from literature,
we predict a role of this motif in protein-protein interaction.
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Building Quantitative Relationship Between Changed
Sequenc and Changed Oxygen Affinity in Human Hemoglobin β-Chain
G. Wu and S. Yan
244 point mutations have been recorded in human hemoglobin
β-chain,
of which some change the oxygen affinity of human hemoglobin
β-chain.
We use the amino-acid distribution probability to quantify
these mutations, and use the cross-impact analysis with Bayes’
law to determine the probability that changes the oxygen affinity
of human hemoglobin β-chain
under mutations.
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Characterization of Fluoroalcohols-Induced Intermediates
of Mucor miehei Lipase at Low pH
S. Fatima, A. Mishra, P. Sen and R.H. Khan
We have previously characterized an acid-unfolded (UA)
state of Mucor miehei lipase at pH 2. The
effect of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol
(HFIP) resulted in characterization of molten-globule (MG)
like states with β-sheet
secondary structure at 15% (v/v) TFE and 6% (v/v) HFIP. α-Helical
states accumulate at 80% (v/v) TFE and 30% (v/v) HFIP.
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Synthesis of Two Peptide Mimetics as Markers for Chemical
Changes of Wool’s Keratin During Skin Unhairing Process
D. Danalev, M. Koleva, D. Ivanova, L. Vezenkov
and N. Vassilev
The sheep skins unhairing process with preliminary alkaline
treatment of the wool leads to two unnatural dipeptide mimetics
lysinoalanine (Lys* - Ala) and ornithinoalanine (Orn*- Ala)
obtaining. They are result from the keratin hydrolysis process.
The changes of wool keratin make it resistant to sulphide
degradation. We synthesized and characterized these unnatural
dipeptides under the experimental conditions. The structures
and mechanism of Lys* - Ala and Orn*- Ala obtaining were elucidated.
The using of newly synthesized products as markers for control
of wool’s keratin changes during skin unhairing process
was demonstrated. The developments have also been the result
of economic and environmental pressures to meet environmental
regulations.
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Synthesis and Biological Evaluation of N-Phosphoryl
Dipeptide Derivatives As Potent Apoptosis Inducers
X. Yu, C. Tan, N. Zhang, W. Xu and Y. Jiang
A series of N-phosphoryl dipeptide derivatives with trimethoxyaniline
moiety were synthesized, in which compound 2a
DIPP-Val-Phe-Ar exhibited the best inhibitory activity against
K562 cells with the IC50
at 9.7 μM.
Its antitumor effects are due to the induction of apoptosis
which was further confirmed by morphological study and flow
cytometry analysis.
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The Interaction Between Cholesterol and Human
Serum Albumin
L. Peng, H. Minbo, C. Fang, L. Xi and Z. Chaocan
The interaction between cholesterol and Human Serum Albumin
(HSA was studied by fluorescence technique. Addition of cholesterol
causes decreasing of the fluorescence intensity of HSA and
the mechanism can be attributed to static quenching. Both
negative enthalpy and entropy change indicate this binding
was an “enthalpy-driven” reaction. The number
of binding site and distance between residues and ligands
were also calculated: n=0.98, r=3.84nm. UV–vis spectra
showed HSA molecules unfolded to some extent and the hydrophobicity
was decreased in the presence of cholesterol.
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Equilibrium Unfolding Mechanism of Chicken Muscle
Triose Phosphate Isomerase
Y. Shi, J.-h. Liu, H.-j. Zhang and Y. Ding
Triose phosphate isomerase (TIM) was prepared and purified
from chicken breast muscle. The equilibrium unfolding of TIM
by urea was investigated by following the changes of intrinsic
fluorescence and circular dichroism spectroscopy, and the
equilibrium thermal unfolding by differential scanning calorimetry
(DSC). Results show that the unfolding of TIM in urea is highly
cooperative and no folding intermediate was detected in the
experimental conditions used. The thermodynamic parameters
of TIM during its urea induced unfolding were calculated asΔG
γ =3.54 kcal·mol-1,
and mG = 0.67 kcal·mo-1·M-1,
which just reflect the unfolding of dissociated folded monomer
to fully unfolded monomer transition, while the dissociation
energy of folded dimer to folded monomer is probe silence.
DSC results indicate that TIM unfolding follows an irreversible
two-state step with a slow aggregation process. The cooperative
unfolding ratio, ΔHca l
/ΔHvH, was measured
close to 2, indicating that the two subunits of chicken muscle
TIM unfold independently. The van’t Hoff enthalpy,
ΔHvH, was estimated
as about 200 kcal·mol-1.
These results support the unfolding mechanism with a folded
monomer formation before its tertiary structure and secondary
structure unfolding.
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Effect of Cosolvents on the Stabilization of Bioactive
Peptides from Bovine Milk α-Casein
S. Srinivas and V. Prakash
Peptides with more than one biological activity are many
a times multifunctional peptides. Two peptides with multifunctional
properties from αS2-casein
were stabilized in presence of cosolvents for their biological
activities like ACE inhibition activity and antioxidant activity.
These bioactive peptides in cosolvents were also thermostable.
Infra red spectra of peptides in cosolvents reveal no change
in the secondary structure in presence of cosolvents. Correlation
between sequence, structure and composition of peptides on
biological activities were studied.
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Purification and Characterization of a Novel Peroxidase
from Bitter Gourd (Momordica charantia)
A. Fatima and Q. Husain
Peroxidase from bitter gourd was purified by three step
purification scheme; ammonium sulphate fractionation, gel
filtration and affinity chromatography. The enzyme was purified
42 fold with the retention of 67% of the initial activity.
The enzyme exhibited its maximum activity at pH 5.6 and 40
oC. The enzyme retained half of its activity even
after 1 h incubation at 60 oC. Molecular weight
of the purified glycosylated bitter gourd peroxidase determined
by Sephacryl S100 and SDS-PAGE was 43 kDa. The stokes radius,
diffusion coefficient and sedimentation coefficient of the
purified peroxidase were 27.3 Å
8.17 x 10-7 cm2/sec and 3.74 S, respectively.
Km for o-dianisidine
and ABTS were 1.3 and 4.9 mM, respectively. The activity of
the enzyme was inhibited by sulfide, azide and L-cysteine.
The carbohydrate content and sulfydryl groups of the enzyme
were 25% (w/w) mass of the protein and 16 mmoles/mole of the
protein, respectively.
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Advanced Glycation: Implications in Tissue Damage
and Disease
A. Gasser and J.M. Forbes
Advanced glycation end products (AGEs) are formed from
the non enzymatic reaction between reducing sugars and amine
residues on proteins, lipoproteins or nucleic acids. AGEs
are found on long-lived proteins and their tissue accumulation
is associated with normal ageing. The formation of AGEs can
be accelerated in certain pathological conditions such as
diabetes where hyperglycaemia is present. AGE modification
of proteins can lead to alterations of normal function by
binding to intracellular or extracellular cell components,
or through receptor binding. This consequently can initiate
a cascade of events, which includes the activation of signal
transduction pathways, which activate inflammatory responses
causing tissue damage. Such tissue injury contributes to the
development of microvascular complications and is of particular
relevance in diabetes where interventions to reduce the accumulation
of AGEs is desirable.
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Using the Concept of Chou’s Pseudo Amino Acid
Composition to Predict Apoptosis Proteins Subcellular Location:
An Approach by Approximate Entropy
X. Jiang, R. Wei, T. Zhang and Q. Gu
The function of protein is closely correlated with it
subcellular location. Prediction of subcellular location of
apoptosis proteins is an important research area in post-genetic
era because the knowledge of apoptosis proteins is useful
to understand the mechanism of programmed cell death. Compared
with the conventional amino acid composition (AAC), the Pseudo
Amino Acid composition (PseAA) as originally introduced by
Chou can incorporate much more information of a protein sequence
so as to remarkably enhance the power of using a discrete
model to predict various attributes of a protein. In this
study, a novel approach is presented to predict apoptosis
protein solely from sequence based on the concept of Chou’s
PseAA composition. The concept of approximate entropy (ApEn),
which is a parameter denoting complexity of time series, is
used to construct PseAA composition as additional features.
Fuzzy K nearest neighbor (FKNN) classifier is selected as
prediction engine. Particle swarm optimization (PSO) algorithm
is adopted for optimizing the weight factors which are important
in PseAA composition. Two datasets are used to validate the
performance of the proposed approach, which incorporate six
subcellular location and four subcellular locations, respectively.
The results obtained by jackknife test are quite encouraging.
It indicates that the ApEn of protein sequence could represent
effectively the information of apoptosis proteins subcellular
locations. It can at least play a complimentary role to many
of the existing methods, and might become potentially useful
tool for protein function prediction. The software in Matlab
is available freely by contacting the corresponding author.
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Identification of a Short Basic Peptide Motif Able
to Drive Copy Number Dependent Nuclear Accumulation of a Linked
Protein
C. Christophe-Hobertus and D. Christophe
The repetitive [RTRG]6
peptide was fortuitously identified as a potent nuclear localization
signal when linked to the green fluorescent reporter protein.
Replacing the arginines by lysines, or the threonines by glycines,
both resulted in a decreased nuclear targeting ability of
the peptide within this context. By contrast, the sequence
[RT]12 proved able to drive
nuclear accumulation of the linked protein as efficiently
as the starting peptide. Remarkably, [RTRG]n
peptides where n=2 to 6 showed a gradual, copy-number dependent,
increase in their ability to target the green fluorescent
protein to the cell nucleus. As a consequence, the nuclear
to cytoplasmic concentration ratio of the linked protein within
the cell could be adjusted to different values depending on
the number of repeats used in the fusion. Our observation
may open the way to the use of [RTRG]n
repeats of given lengths (n=2 to 6) for fixing the nuclear
cytoplasmic partition of shuttling protein domains in the
course of their functional study.
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Production, Purification and Characterization of β-1,4
Endoglucanase from a Novel Bacterial Strain CTP-09 of a Bacillu
sp.
M. Saleem, M.S. Akhtar, R. Yasmin, M. Zahid, N.N. Malik,
M. Afzal and M.I. Rajoka
Bacillus strain CTP-09 yielded maximum productivity
(1120 IU/L.h) of extracellular endoglucanase (CMCase) on 0.5%
cellobiose after 10 h fermentation at 55o C. The
purified enzyme is mono-meric in nature and exhibits stability
up to 80o C and over a pH range (6.0-9.0). Activation
energy, enthalpy and entropy of catalysis, and inactivation
indicated that this CMCase is highly thermos-table. Purified
enzyme possessed high power of defibrillation of textile and
was minutely inhibited by anionic detergent and oxidizing
agent comparable with inhibition by commercial enzyme. This
polypeptide could be exploited for mass production and application
in local industries.
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Crystallization and Preliminary X-Ray Diffraction Analysis
of a Novel Mannose-Binding Lectin with Antiretroviral Properties
from Polygonatum cyrtonema Hua
J.-J. Ding, J.-k. Bao, D.-Y. Zhu, Y. Zhang and
D.-C. Wang
A novel antiretroviral protein Polygonatum cyrtonema
lectin (PCL) belonging to the monocot mannose-binding lectin
(MMBL) superfamily has been crystallized using hanging-drop
vapor-diffusion method. The crystals diffract to 2.0 Å
resolution and belong to space group P21,
with unit-cell parameters of a=39.308 Å,
b=48.317 Å,
c=112.221 Å,
and β=90.12°.
Preliminary analysis indicates that the asymmetric unit contains
four PCL molecules with a solvent content of about 45%. A
set of X-ray data has been collected for the crystal structure
determination.
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Crystallization and Preliminary X-Ray Diffraction
Studies of a Psychrophilic Iron Superoxide Dismutase from
Pseudoalteromonas haloplanktis
A. Merlino, I.R. Krauss, I. Castellano,
E. De Vendittis, A. Vergara and F. Sica
The Antarctic eubacterium Pseudoalteromonas haloplanktis
(Ph) produces a cold-active iron superoxide
dismutase (SOD). PhSOD is a homodimeric enzyme, that
displays a high catalytic activity even at low temperature.
Using hanging-drop vapour-diffusion technique, PhSOD
has been successfully crystallized in two different crystal
forms. Both crystal forms are monoclinic with space group
P21 and diffract to 2.1 Å
resolution. Form I has unit-cell parameters a=45.49Å
b=103.63Å
c=50.37Å
β=108.2°
and contains a homodimer in the asymmetric unit. Form II has
unit-cell parameters a=50.48Å
b=103.78Å
c=90.25Å
β=103.8° and an asymmetric unit containing
two PhSOD homodimers. Structure determination has
been achieved using molecular replacement. The crystallographic
study of this cold-adapted enzyme could contribute to the
understanding of the molecular mechanisms of cold-adaptation
and of the high catalytic efficiency at low temperature.
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Crystallization and Preliminary X-Ray Crystallographic
Analysis of Galectin LEC-1 from Caenorhabditis elegans
T. Itagaki, S. Nishizaki, K. Sekihashi, H. Kobayashi,
S.-i. Kidokoro, Y. Kezuka, Y. Arata, J. Hirabayashi, K.-i.
Kasai and T. Nonaka
Galectin LEC-1 isolated from the nematode Caenorhabditis
elegans was the first galectin found in invertebrates
and also the first tandem-repeat type galectin identified,
containing two homologous carbohydrate binding sites. This
galectin is localized most abundantly in the adult cuticle
and possibly plays a role in the formation of epidermal layers.
We succeeded in crystallizing LEC-1 composed of 279 amino
acids with a calculated molecular weight of 31,809 Da under
two independent sets of conditions as a result of extensive
screening. The crystals grown under one set of conditions
belong to the triclinic space group P1, with unit-cell
parameters α
= 48.44, b = 52.13, c = 64.24Å,
α = 108.73, β=
91.39 , and γ
= 98.45° and two protein molecules per unit cell. The
crystals grown under the other set of conditions which included
lactose belong to the monoclinic space group P21
, with unit-cell parameters α
= 52.90, b = 47.01, c = 66.16 Å,
and β=
113.30° and one protein molecule per asymmetric unit.
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Purification, Crystallization and Preliminary
Crystallographic Analysis of CYP 195A2, a P450 Enzyme from
Rhodopseudomonas palustris
D. Guo, F. Xu, S.G. Bell, X. Pang, M. Bartlam and
L.-L. Wong
Cytochrome P450 monooxygenases are a superfamily of heme-thiolate
proteins involved in the metabolism of a wide variety of endogenous
and xenobiotic compounds. The P450 enzyme CYP195A2 from Rhodopseudomonas
palustris CGA009, a metabolically versatile bacterium,
was overproduced in E. coli and purified. Two distinct
crystal forms were obtained under separately optimized conditions
by the hanging-drop vapor-diffusion method. Native data sets
extending to resolutions of 2.3 Å
and 2.8 Å
have been collected and processed in space groups P222 and
C2221 respectively.
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