| Protein
& Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 15, Number 6, 2008
Contents
Regular Papers
Solvent Modulation of Column Chromatography
Pp. 544-555
T. Arakawa, Y. Kita, D. Ejima and P. Gagnon
[Abstract]
Intra-Molecular Electron Transfer in Proteins
Pp. 556-561
T. Brittain
[Abstract]
Solubility Improvement of an Anthrax Toxin Peptide
Inhibitor by Rational Amino Acid Randomization Pp.
562-566
A. Pini, J. Brunetti, C. Falciani, M. Fabbrini and L.
Bracci
[Abstract]
Proteomic Analysis of Peanut Seed Storage Proteins
and Genetic Variation in a Potential Peanut Allergen
Pp. 567-577
B. Guo, X. Liang, S.-Y. Chung, C.C. Holbrook and S.J.
Maleki
[Abstract]
Insects Antiviral and Anticancer Peptides: New
Leads for the Future? Pp. 578-585
M. Slocinska, P. Marciniak and G. Rosinski
[Abstract]
Role of Polyglycine Repeats in the Regulation
of Glycogen Synthase Kinase Activity Pp. 586-589
F. Hernández, A. Gómez-Ramos, P. Goñi-Oliver,
J. Avila and N. Villanueva
[Abstract]
Predicting Membrane Protein Types with Bagging
Learner Pp. 590-594
B. Niu, Y.-H. Jin, K.-Y. Feng, L. Liu, W.-C. Lu, Y.-D.
Cai and G.-Z. Li
[Abstract]
Extracellular Proteome Changes of Deinococcus
radiodurans Under γ-Irradiation
Stress Conditions Pp. 595-599
N. Ying, Z. Zheng, H. Xu, B. Tian and Y. Hua
[Abstract]
Biochemical Characterization of Two DNA Ligases
from Deinococcus radiodurans Pp. 600-605
D. Le, X. Hua, L. Huang, G. Gao, H. Lu, Z. Xu, B. Tian
and Y. Hua
[Abstract]
Pea Lectin in Alkaline Conditions: Formation
of Molten Globule Like Intermediate and Its Structural and
Thermal Studies Under the Influence of Hexafluoroisopropanol
Pp. 606-611
F. Naseem and R.H. Khan
[Abstract]
Predicting Protein Subcellular Location Using
Chou’s Pseudo Amino Acid Composition and Improved Hybrid
Approach Pp. 612-616
F.-M. Li and Q.-Z. Li
[Abstract]
Characterization of Mutants of Sulfolobus
solfataricus Signature Amidase Able to Hydrolyze R-Ketoprofen
Amide Pp. 617-623
C. Giordano and S. Ammendola
[Abstract]
The 5S Subunit of Transcarboxylase Interacts
with Free Biotin as Studied by Transferred-NOESY and Saturation
Transfer Difference NMR Pp. 624-329
R.K. Bhat and S. Berger
[Abstract]
A Preliminary X-Ray Study of a Refolded PTS EIIBfruc
Protein from Escherichia coli Pp. 630-632
D.H. Shin
[Abstract]
Comparative Studies on the Aggregation Behavior
of HBPs from Human Seminal Plasma by Dynamic Light Scattering
Pp. 633-639
V. Kumar, Md. I. Hassan, T.P. Singh and S. Yadav
[Abstract]
Putative Secondary Active Site of Bovine Pancreatic
Deoxyribonuclease I Pp. 640-646
W.-J. Chen, P.-T. Huang, Y-C. Cheng and T.-H. Liao
[Abstract]
Crystallization Report
Crystallization and Preliminary X-Ray Analysis of
the Splice Variant of Human Ankyrin Repeat and Suppressor
of Cytokin Signaling Box Protein 9 (hASB9-2) Pp.
647-649
X. Fei, Y. Zhang, X. Gu, R. Qiu, Y. Mao and C. Ji
[Abstract]
Abstracts
[Back to top]
Solvent Modulation of Column Chromatography
T. Arakawa, Y. Kita, D. Ejima and P. Gagnon
A majority of column chromatographies use only selected
salts, e.g., ammonium sulfate, NaCl, Citrate and phosphate
in hydrophobic interaction chromatography (HIC) and NaCl in
ion exchange and dye affinity chromatographies. Alternatively,
a pH range below or above the neutral value is often used
to reduce affinity interactions, e.g., in Protein-A or dye
affinity column chromatography. Although these parameters
are easily manipulated, they are not necessarily the optimal
conditions for high recovery and resolution of the proteins.
So-called co-solvents have been used, although to a limited
extent, to manipulate performance of column chromatography.
Here the term co-solvent is used to indicate its relatively
high concentrations required for these applications, meaning
that it also serves as solvent along with water. Ethylene
glycol and MgCl2 have been
used to elute specific antibodies from antigen-affinity column.
Arginine has also been used for the same purpose. Arginine
has much wider applications for various column chromatographies,
including size exclusion chromatography (SEC), HIC and affinity
chromatography. Polyethylene glycol and glycine have also
been used to improve the performance of HIC and hydroxyapatite
chromatography. This review summarizes these applications
of co-solvents for column chromatographies.
[Back to top]
Intra-Molecular Electron Transfer in Proteins
T. Brittain
Intra-molecular electron transfer is a key process, which
is of prime importance, in photosynthesis, mitochondrial electron
transfer and the action of many multi-centre enzymes. This
mini-review considers the possible mechanisms of intra-molecular
electron transfer in proteins and reviews the recent developments
relating to possible electron tunnelling and electron hopping
processes within di-heme cytochrome c peroxidase.
[Back to top]
Solubility Improvement of an Anthrax Toxin Peptide Inhibitor
by Rational Amino Acid Randomization
A. Pini, J. Brunetti, C. Falciani, M. Fabbrini and L.
Bracci
We previously described a potent anthrax toxin inhibitor,
based on a phage-library-selected peptide sequence, synthesized
as a tetra-branched molecule on a lysine core and further
modified for improvement of activity [Pini et al.,
Biochem. J., 2006, 395, 157]. This branched peptide
had very low solubility because of several hydrophobic residues
in the peptide sequence. This complicated molecule purification
and manufacturing. Here we report a rational modification
of the peptide sequence, obtained by construction and selection
of several mini libraries of branched peptides, containing
sequences randomized in non crucial positions of the original
peptide. Mini libraries were screened for solubility and inhibitory
activity. This procedure enabled us to obtain a new peptide
with a better solubility and identical inhibitory activity
[Back to top]
Proteomic Analysis of Peanut Seed Storage Proteins and Genetic
Variation in a Potential Peanut Allergen
B. Guo, X. Liang, S.-Y. Chung, C.C. Holbrook and S.J.
Maleki
Peanut allergy is one of the most severe food allergies.
One effort to alleviate this problem is to identify peanut
germplasm with lower levels of allergens which could be used
in conventional breeding to produce a less allergenic peanut
cultivar. In this study, we identified one peanut line, GT-C9,
lacking several seed proteins, which were identified as Ara
h 3 isoforms by peptide sequencing and named iso-Ara h 3.
Total seed proteins were analyzed by one-dimensional (SDS-PAGE)
and two-dimensional gel electrophoreses (2-D PAGE). The total
protein extracts were also tested for levels of protein-bound
end products or adducts such as advanced glycation end products
(AGE) and N-(carboxymethyl) lysine (CML), and IgE
binding. Peanut genotypes of GT-C9 and GT-C20 exhibited significantly
lower levels of AGE adducts and of IgE binding. This potential
peanut allergen iso-Ara h 3 was confirmed by peptide
sequences and Western blot analysis using specific anti-Ara
h 1, Ara h 2, and Ara h 3 antibodies. A full-length sequence
of iso-ara h 3 (GenBank number DQ855115) was obtained. The
deduced amino acid sequence iso-Ara h 3 (ABI17154) has the
first three of four IgE-binding epitopes of Ara h 3. Anti-Ara
h 3 antibodies reacted with two groups of protein peptides,
one with strong reactions and another with weak reactions.
These peptide spots with weak reaction on 2-D PAGE to anti-Ara
h 3 antibodies are subunits or isoallergens of this potential
peanut allergen iso-Ara h 3. A recent study suggested that
Ara h 3 basic subunits may be more significant allergenicity
than the acidic subunits.
[Back to top]
Insects Antiviral and Anticancer Peptides: New Leads for the
Future?
M. Slocinska, P. Marciniak and G. Rosinski
Insect produce wide range of protein and peptides as
a first fast defense line against pathogen infection. These
agents act in different ways including insect immune system
activation or by direct impact on the target tumor cells or
viruses. It has been shown that some of the insect peptides
suppress viral gene and protein expression, rybosilate DNA,
whereas others cause membrane lysis, induce apoptosis or arrest
cell cycle. Several of the purified and characterized peptides
of insect origin are very promising in treating of serious
human diseases like human immunodeficiency virus (HIV), herpex
simplex virus (HSV) or leukaemia. However, some obstacles
need to be overcome. Cytotoxic activity of peptides, susceptibility
to proteases or high cost of production remain still unsolved
problems.
Reports on the peptides antiviral and antitumour mechanisms
are scanty. Thus, in this review we present characteristic,
mode of action and potential medical applications of insects
origin peptides with the antiviral and antitumour activity.
[Back to top]
Role of Polyglycine Repeats in the Regulation of Glycogen
Synthase Kinase Activity
F. Hernández, A. Gómez-Ramos, P. Goñi-Oliver,
J. Avila and N. Villanueva
Glycogen synthase kinase (GSK3) activity present in one
cell is the consequence of the sum of the activities of two
different proteins called GSK3α
and GSK3β.
These isoenzymes are coded by two different genes and share
an almost identical sequence at their catalytic domain, but
differ in the sequence of putative regulatory regions. In
this review, we propose that glycine repeats present only
in GSK3α
may result in the different cleavage of both isoenzymes by
the protease calpain, a cleavage that modifies GSK3 activity.
[Back to top]
Predicting Membrane Protein Types with Bagging Learner
B. Niu, Y.-H. Jin, K.-Y. Feng, L. Liu, W.-C. Lu, Y.-D.
Cai and G.-Z. Li
The membrane protein type is an important feature in
characterizing the overall topological folding type of a protein
or its domains therein. Many investigators have put their
efforts to the prediction of membrane protein type. Here,
we propose a new approach, the bootstrap aggregating method
or bragging learner, to address this problem based on the
protein amino acid composition. As a demonstration, the benchmark
dataset constructed by K.C. Chou and D.W. Elrod was used to
test the new method. The overall success rate thus obtained
by jackknife cross-validation was over 84%, indicating that
the bragging learner as presented in this paper holds a quite
high potential in predicting the attributes of proteins, or
at least can play a complementary role to many existing algorithms
in this area. It is anticipated that the prediction quality
can be further enhanced if the pseudo amino acid composition
can be effectively incorporated into the current predictor.
An online membrane protein type prediction web server developed
in our lab is available at http://chemdata.shu.edu.cn/protein/protein.jsp.
[Back to top]
Extracellular Proteome Changes of Deinococcus radiodurans
Under γ-Irradiation
Stress Conditions
N. Ying, Z. Zheng, H. Xu, B. Tian and Y. Hua
To analysis the change of Deinococcus radiodurans
extracellular proteins recovering from γ-irradiation,
we examined extracellular proteome changes using two-dimensional
polyacrylamide gel electrophoresis. Twenty-six spots on the
gel of irradiated sample were showed significant changes compared
with spots on the control gel. Using peptide mass fingerprinting
via matrix-assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF-MS), 21 different proteins could
be distinguished. Among the identified proteins, seven are
classified in transport and metabolism, and one is involved
in intracellular trafficking and secretion. The other proteins
are known to several functions in the cytosol. Most of the
proteins have not previously been reported to be relevant
to radioresistance. These results imply that the transmembrane
transportation is involved in and contributes to the radioresistance
in this organism.
[Back to top]
Biochemical Characterization of Two DNA Ligases from Deinococcus
radiodurans
D. Le, X. Hua, L. Huang, G. Gao, H. Lu, Z. Xu, B. Tian
and Y. Hua
Two genes encoding a NAD+ -dependent
DNA ligase (LigA) and an ATP-dependent DNA ligase (LigB) were
identified in the genome of the extremely radioresistant bacterium,
Deinococcus radiodurans (DR). The recombinant
enzymes expressed in Escherichia coli, were purified
to homogeneity and characterized. The optimal temperature
and pH value of the two DNA ligases were 60 oC and 7.0, respectively.
Their optimal concentration of MgCl2
was 5mM. Their half-lifes of heat inactivation at 100 oC were
about 3 min and 5 min, respectively. In addition, the results
showed that DRLigB displayed higher activity than DRLigA at
stick and blunt ended joining of DNA, indicating that DRLigB
is a key DNA ligase of D. radiodurans in DNA recombination
and double-strand break repair.
[Back to top]
Pea Lectin in Alkaline Conditions: Formation of Molten Globule
Like Intermediate and Its Structural and Thermal Studies Under
the Influence of Hexafluoroisopropanol
F. Naseem and R.H. Khan
Pea lectin was exposed to a pH range of 7-13. It was
observed that alkaline-unfolding resulted in a molten globule-like
intermediate at pH 11. The structural stability of this alkaline
unfolded molten globule-like state of pea lectin was studied
in the presence of HFIP. Thermal studies showed that this
state was more susceptible to thermal denaturation as compared
to the native state and it became even more so in presence
of HFIP.
[Back to top]
Predicting Protein Subcellular Location Using Chou’s
Pseudo Amino Acid Composition and Improved Hybrid Approach
F.-M. Li and Q.-Z. Li
The location of a protein in a cell is closely correlated
with its biological function. Based on the concept that the
protein subcellular location is mainly determined by its amino
acid and pseudo amino acid composition (PseAA), a new algorithm
of increment of diversity combined with support vector machine
is proposed to predict the protein subcellular location. The
subcellular locations of plant and non-plant proteins are
investigated by our method. The overall prediction accuracies
in jackknife test are 88.3% for the eukaryotic plant proteins
and 92.4% for the eukaryotic non-plant proteins, respectively.
In order to estimate the effect of the sequence identity on
predictive result, the proteins with sequence identity =40%
are selected. The overall success rates of prediction are
86.2% and 92.3% for plant and non-plant proteins in jackknife
test, respectively.
[Back to top]
Characterization of Mutants of Sulfolobus solfataricus
Signature Amidase Able to Hydrolyze R-Ketoprofen Amide
C. Giordano and S. Ammendola
The amidase from Sulfolobus solfataricus enantioselectively
hydrolyzes S-ketoprofen amide to its corresponding acid. We
identify three independent SsAH mutants that hydrolyze R-ketoprofen
amide and built computational models of their three-dimensional
structure. Interestingly the mutations do not specifically
affect residues near the active site, or directly interacting
with the substrate.
[Back to top]
The 5S Subunit of Transcarboxylase Interacts with Free Biotin
as Studied by Transferred-NOESY and Saturation Transfer Difference
NMR
R.K. Bhat and S. Berger
The 5S subunit of transcarboxylase was expressed and
purified. Recent methods of NMR spectroscopy as transferred
NOESY, INPHARMA and Saturation Transfer Difference (STD) NMR
were used to investigate ligand binding of free biotin to
the 5S protein. The binding epitope for biotin is very similar
to that obtained at the 12S subunit of tran-scarboxylase,
however no common binding site for pyruvate and biotin exists.
[Back to top]
A Preliminary X-Ray Study of a Refolded PTS EIIBfruc
Protein from Escherichia coli
D.H. Shin
The phosphoenolpyruvate-carbohydrate phosphotransferase
system (PTS) catalyzes the phosphorylation and transportation
of its sugar substrates. A sugar-specific enzyme II complex
involved in the PTS finally functions to translocate substrates
across the membrane. A PTS EIIBfruc
protein, a fructose specific EIIB subunit, from Escherichia
coli has been cloned, expressed, refolded, purified,
and crystallized. The synchrotron data were collected to 2.6
Å from the crystal of a selenomethionine substitute
PTS EIIBfruc protein. The
crystal belongs to the primitive trigonal space group P3121,
with unit-cell parameters of a = 33.4 Å, b
= 33.4 Å, c = 154.0 Å, and β
= 120.0 γ.
A full structure determination is under way to provide insights
into the structure-function relationships of this protein.
[Back to top]
Comparative Studies on the Aggregation Behavior of HBPs from
Human Seminal Plasma by Dynamic Light Scattering
V. Kumar, Md. I. Hassan, T.P. Singh and S. Yadav
Heparin binding proteins (HBPs) from human seminal plasma
(HSP) were obtained by heparin affinity and size exclusion
chromatography. The aggregation/disaggregation of HBPs was
followed by dynamic light scattering (DLS) in presence of
various physiological ligands such as CaCl2
, NaCl, EDTA, cholesterol, adenosine, D-glucose and D-fructose.
The aggregation pattern of lactoferrin was also analyzed and
compared. The study of interactions of HBPs of HSP may contribute
to an understanding mechanisms underlying the fertilization
process.
[Back to top]
Putative Secondary Active Site of Bovine Pancreatic Deoxyribonuclease
I
W.-J. Chen, P.-T. Huang, Y-C. Cheng and T.-H. Liao
Previous structural studies based on the co-crystal of
a complex between bovine pancreatic deoxyribonuclease I (bpDNase
I) and a double-stranded DNA octamer d(GCGATCGC)2
have suggested the presence of a putative secondary active
site near Ser43. In our present study, several crucial amino
acid residues postulated in this putative secondary active
site, including Thr14, Ser43, and His44 were selected for
site-directed mutagenesis. A series of single, double and
triple mutants were thus constructed and tested for their
DNase I activity by hyperchromicity assay. Substitution of
each or both of Thr14 and Ser43 by alanine results in mutant
enzymes retaining 30-70% of WT bpDNase I activity. However,
when His44 was replaced by aspartic acid, either in the single,
double, or triple mutant, the enzyme activities were drastically
decreased to 0.5-5% that of WT bpDNase I. Interestingly, when
cysteine was substituted for Thr14 or Ser43, the specific
DNase activities of the mutant enzymes were substantially
increased by 1.5-100-fold, comparing to their alanine substitution
mutant counterparts. Two other more sensitive DNase activity
assay method, plasmid scission and zymogram analyses further
confirm these observations. These results suggested that His44
may play a critical role in substrate DNA binding in this
putative secondary active site, and introduction of sulfhydryl
groups at Thr14 and Ser43 may facilitate Mn2+
-coordination and further contribute to the catalytic
activity of bpDNase I.
[Back to top]
Crystallization and Preliminary X-Ray Analysis of
the Splice Variant of Human Ankyrin Repeat and Suppressor
of Cytokine Signaling Box Protein 9 (hASB9-2)
X. Fei, Y. Zhang, X. Gu, R. Qiu, Y. Mao and C. Ji
Human ankyrin repeat and suppressor of cytokine signaling
box protein 9 (hASB9), a subunit of an Elongin C-cullin-SOCS
box (ECS) E3 ubiquitin ligase complex, is believed to be involved
in specific substrate-recognition for ubiq-uitination and
degradation. In fact, this specific substrate-recognition
is determined by the ankyrin repeats of hASB9 protein. Here,
we have cloned and overexpressed the hASB9-2, the splice variant
of hASB9 with only one ankyrin repeat domain, as a 6His-tagged
recombinant protein in Escherichia coli. The purified
hASB9-2 protein was crystallized by the hanging-drop vapor-diffusion
technique and diffracted to 2.2Å resolution. The data
showed that the cubic hASB9-2 crystal belongs to space group
P4332 with unit-cell parameters
(a=b=c=129.25Å, α=β=γ=90°).
An asymmetric unit in the crystal was assumed to contain one
protein molecule giving the Matthews Coefficient factor of
2.81 and the solvent content of 56.3%.
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