Contents
Rational
and Random Strategies for the Mimicry of Discontinuous Protein Binding Sites Pp.281-290
Design,
Synthesis and Antibacterial Activity Studies of Model Peptides of Proline/
Arginine-Rich Region in Bactenecin7 Pp.291-300
K.
Abiraj, H.S. Prasad, A.S. Prakasha Gowda and D. Channe Gowda
Direct
Comparison of the Crystal and Solution Structure of Xylanase from Trichoderma
longibrachiatum Pp.301-306
Maciej
Kozak
Alkali-Induced
Conformational Transition in Different Domains of Bovine Serum Albumin Pp.307-315
Basir
Ahmad, Mohammad Zahid Kamal and Rizwan Hasan Khan
Production
of Recombinant Human Osteoprotegrin from Trichoplusia ni Cells and Bombyx mori
Larvae Pp.317-323
Zhen
Liu, Guan-zhen Yang and Xiang-Fu Wu
Volumetric Behavior of the Molten
Globule State of Canine Milk Lysozyme Pp.325-330
Masahiro
Watanabe, Yoshihiro Kobashigawa, Tomoyasu Aizawa, Makoto
Demura and Katsutoshi Nitta
Binding of Lead Ion to Bovine
Serum Albumin Studied by Ion Selective Electrode Pp.331-337
Erol
Ayranci, Osman Duman
Preferential Codons Enhancing the
Expression Level of Human Beta-Defensin-2 in Recombinant Escherichia coli Pp.339-344
Li
Peng , Zhinan Xu, Xiangming Fang, Fang Wang, Sheng Yang and Peilin Cen
An Opaque-2 Like Transcription
Factor from Pearl Millet Pp.345-352
Lucilia
Helena Marcellino, Goran Neshich, Antonio Furtado Neto and Eugen Silvano Gander
Chemically Modified Porcine
Hemoglobins and Their Biological Properties Pp.353-360
Cai
Jin, Meng Wen-fang, Hong Min, Cen Pei-lin and Yuan Zhong-yi
Specific and Non-Specific Contacts in Protein Crystals Pp.361-366
Feng
Dan and Zeng Zong-Hao
Complementary Peptide Epitopes
and Anti-Idiotypic Antibodies in Autoimmunity Pp.367-375
Maria
Sakarellos-Daitsiotis, Manh-Thong Cung, Constantinos Sakarellos, Zouhair El
Hilali, Aggeliki Kosmopoulou and Chryssa Voitharou
Amyloid-b as a “Difficult Sequence” in
Solid Phase Peptide Synthesis Pp.377-384
Anna
K. Tickler, Andrew B. Clippingdale and John D. Wade
Influenza A Hemagglutinin
C-Terminal Anchoring Peptide: Identification and Mass Spectrometric Study Pp.385-391
Larisa
V. Kordyukova, Aleksander L. Ksenofontov, Marina V. Serebryakova, Tatyana V.
Ovchinnikova, Natalija V. Fedorova, Valeria T. Ivanova and Ludmila A. Baratova
Crystallization and Preliminary
X-Ray Analysis of Complexes of Porcine Pancreatic Elastase with Two Natural
Inhibitors Pp.393-399
Kai
Hilpert, Helga Wessner, Christa Scholz, Patrick Scheerer, Rudolf Volkmer-Engert
and Norbert Krauß
Crystallization and Preliminary
X-Ray Analysis of Plant Class I Chitinase from Rice Pp.401-405
Yuichiro
Kezuka, Keiji Kitazaki, Yoshikane Itoh, Jun Watanabe, Osamu Takaha, Takeshi
Watanabe, Yoko Nishizawa and Takamasa Nonaka
Abstracts
[Back to top] Rational
and Random Strategies for the Mimicry of Discontinuous Protein Binding Sites
Jutta
Eichler
The high technical
standard of current peptide chemistry, which has evolved over the past three decades,
has profoundly facilitated the investigation of proteins and their interactions
with other molecules at the level of individual amino acids. Using currently
available peptide synthesis methods, sequentially continuous protein binding
sites can be readily mapped, characterized, optimized, and used as lead
compounds for inhibitors of protein-ligand interactions. The mimicry of
sequentially discontinuous protein binding sites, on the other hand, continues
to present a challenge for peptide and organic chemists. This mini-review
summarizes currently used and emerging, rational and random strategies for the
design of synthetic mimetics of discontinuous protein binding sites.
[Back to top]
Design, Synthesis and
Antibacterial Activity Studies of Model Peptides of Proline/ Arginine-Rich
Region in Bactenecin7
K. Abiraj, H.S. Prasad, A.S. Prakasha Gowda and D. Channe Gowda
Bactenecin7
(Bac7), a cationic antibacterial peptide, contains a repeating region of
Xaa-Pro-Arg-Pro (Xaa = hydrophobic residue). To investigate the structure and
property of a Pro/Arg-rich region, we synthesized a series of peptides,
Xaa-Pro-Arg-Pro (Xaa = Gly, Arg, Leu, Ile, and Phe) as models and
characterized. The conformational preferences of these peptides in water and
trifluoroethanol were examined by circular dichroism. The results suggest the
presence of largely poly(Pro)-II helical conformation in aqueous and
trifluoroethanol solutions. Their antibacterial activity against gram-negative
bacteria such as Escherichia coli, Klebsiella Pneumoniae, Pseudomonas
aeruginosa, and Escherichia coliHB101, and grampositive bacteria
such as Staphylococcus aureus were measured at various peptide concentrations.
Two of our synthetic tetrapeptide fragments containing Gly and Arg were
efficiently killed the gram-positive bacteria, Staphylococcus aureus, at the
concentration level of 200 µg/mL.
[Back to top]
Direct Comparison of
the Crystal and Solution Structure of Xylanase from Trichoderma longibrachiatum
Maciej
Kozak
The small angle
X-ray scattering (SAXS) data of xylanase XYNII (endo-1,4-b-xylan xylanohydrolase EC 3.2.1.8) from Trichoderma
longibrachiatum, an enzyme catalysing the reaction of accidental hydrolysis
of b-1,4-D-xylosidic linkages of xylan, were
recorded for protein solution using synchrotron radiation. The experimental
data were compared with those of theoretical scattering calculated on the basis
of the known crystal structure. The radius of gyration measured by SAXS (RG=1.7
nm) was about 3.5% larger and the maximum dimension in the distance
distribution function about 5 % larger than the corresponding values calculated
on the basis of the crystal structure.
[Back to top] Alkali-Induced Conformational Transition in Different
Domains of Bovine Serum Albumin
Basir
Ahmad, Mohammad Zahid Kamal and Rizwan Hasan Khan
Alkaline pH
induced conformational changes in different domains of bovine serum albumin
were studied by using domain specific ligands: chloroform, bilirubin and
diazepam for domains I, II and III respectively. The effect of alkaline pH on
the secondary structure of BSA was monitored by far-UV CD in the range 250 nm
to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step
change, one corresponding to N«B
transition (pH 7.5 to 9.0) and the other to B ® U
(pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing
pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased
thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0
and decreased thereafter reaching a value similar to one obtained with native
BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished
completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in
chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH
9.5 to 11.0) suggested major loss of native conformation of domain I followed
by domain III during alkaline induced unfolding of BSA. However, a significant
increase in bilirubin binding showed a favorable conformational rearrangement
in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete
abolishment of bilirubin binding to BSA and significant loss of secondary structure
around pH 12.5 indicated that domain II was more resistant to alkaline pH and
unfolds only at extreme alkalinity. Taken
together, these
data suggest that unfolding of three domains of BSA follow the following order
of susceptibility towards alkaline denaturation of BSA domain I>domain
III>domain II.
[Back
to top] Production of Recombinant Human
Osteoprotegrin from Trichoplusia ni Cells and Bombyx mori Larvae
Zhen
Liu, Guan-zhen Yang and Xiang-Fu Wu
Human
osteoprotegrin (OPG) and its truncated mutant OPG-280 and lengthened mutant
OPG-Fc were constructed and successfully expressed in Trichoplusia ni
cells and Bombyx mori larvae. Native SDSPAGE and Western blot analysis
revealed that OPG-Fc is present as a homodimer in Tn cells or B. mori
larvae compared with OPG and OPG-280. Furthermore, the hypocalcemic effect
assay showed that truncation of the C-terminal 100 residues OPG does not
abolish the biological activity and Fc can be helpful in forming the OPG
homodimer with improved biological activity.
[Back to top] Volumetric Behavior of the Molten
Globule State of Canine Milk Lysozyme
Masahiro
Watanabe, Yoshihiro Kobashigawa, Tomoyasu Aizawa,Makoto Demura and Katsutoshi Nitta
The effect of pressure
on the unfolding of the molten globule (MG) state of canine milk lysozyme (CML)
was examined using ultraviolet spectroscopy. The volume changes of the
MG-unfolded-state transition were observed at pH 2.0 and around 20 to 60°C, but
no volume change has been found for bovine a-lactalbumin,
which is homologous to CML. Our results suggest that the MG state of CML
possesses a tightly packed hydrophobic core.
[Back to top] Binding of Lead Ion to Bovine Serum
Albumin Studied by Ion Selective Electrode
Erol
Ayranci, Osman Duman
The binding of Pb2+
to bovine serum albumin (BSA) at neutral pH was studied using lead ion
selective electrode. The binding data was treated according to Scatchard
Equation. The number of binding classes and the number of binding sites,
intrinsic dissociation constants and stepwise binding constants for each class
were determined. Two binding classes were found. Four binding sites in the
first class and five binding sites in the second class were determined. Binding
in the first class was stronger than in the second. Similar binding studies
were carried out with heat treated BSA. It was found that not only the number
of binding sites but also the strength of binding increases upon heat
treatment.
[Back to top] Preferential Codons Enhancing the
Expression Level of Human Beta-Defensin-2 in Recombinant Escherichia coli
Li
Peng , Zhinan Xu, Xiangming Fang, Fang Wang, Sheng Yang and Peilin Cen
Human b-defensin-2 (hBD2) is a small antimicrobial
peptide with potential as a therapeutic agent. The effect of codon usage on the
expression of hBD2 in Escherichia coli was studied. Two coding sequences
encoding the same hBD2 precursor were both expressed as fusion protein with
thioredoxin in E. coli BL21 (DE3). One is the wild-type human cDNA and
the other is a gene synthesized by a PCR-based method in which rare codons were
altered to those frequently used in E. coli. The expression level of
recombinant hBD2 was over 50% of the total cellular protein when the synthetic
gene with preferential codons was employed which was a 9-fold enhancement over
the wild-type cDNA. The result shows the codon bias of the host was a major
barrier in high-level expression of recombinant hBD2 and suggests a similar
approach may be used in the expression of other defensins in E. coli.
[Back to top] An Opaque-2 Like Transcription
Factor from Pearl Millet
Lucilia
Helena Marcellino, Goran Neshich, Antonio Furtado Neto and Eugen Silvano Gander
Pearl millet (Pennisetum
glaucum L.), a species of the Poaceae family, is an important food
crop in Africa, Asia and South America. Its nutritional value is due to storage
prolamins accumulated in the seeds. In other species of the same family, the
expression of the genes coding for storage prolamins is mediated by the
regulatory protein opaque-2. In this paper we show that an opaque-2 –like
protein is present in pearl millet too and is expressed during the early stages
of seed development. The organization of the gene coding for this protein is
similar to that of orthologous genes in other Poaceae species, i.e. six
exons separated by five introns. A comparison of amino acid homologies with
other described opaque-2 proteins is presented.
[Back to top] Chemically Modified Porcine
Hemoglobins and Their Biological Properties
Cai
Jin, Meng Wen-fang, Hong Min, Cen Pei-lin and Yuan Zhong-yi
Hemoglobin cross-linked
with small molecular modifiers turns out to be more stable. Modifications of
proteins with polyethylene glycol (PEG) have been proven to enlarge the
molecular size of proteins, to prolong their retention time in the circulation
as well as blunt immune reactions. In the present study, the optimal conditions
for porcine hemoglobin (pHb) modification with bis (3, 5-dibromosalicyl)
fumarate (DBBF) and PEG were evaluated. The derivative of DBBF cross-linked pHb
(DBBF-pHb) showed improved oxygen affinity and the ability to resist the
dissociation of the a2b2 tetramer compared with the natural protein.
DBBF-pHb was then bound to the activated PEG. The results indicated that the
pHb modified with DBBF and PEG had more stable tetrameric conformation with a
molecular weight of 107000. Their oxygen half-saturation pressure (P50)
is around 3.33 kPa, which approximates the physiological P50
of human red blood cells. Both routine and reinforced immunizing methods were
adopted to study the immunogenicity of modified products and the results showed
that the products had very low immunogenicity evaluated by enzyme-linked
immunoadsordent assay (ELISA). Somewhat beneficial effects were shown in the
treatment of hemorrhagic shock where modified hemoglobin solutions were used as
resuscitation fluids in the hemorrhagic shock Sprague-Dawley (SD) rats model.
[Back to top] Specific and Non-Specific
Contacts in Protein Crystals
Feng
Dan and Zeng Zong-Hao
Statistical
analysis of protein-protein interfaces in a database of pure peptide crystals
shows that the distribution of the contact area contains two components: a
major exponential distribution and a minor flat distribution. Analysis of two
sub-databases provides evidence that the two components represent specific and
non-specific contacts, respectively. The probability of an interface with a
given area being specific can be estimated. A scaled quantity (contact ratio)
is introduced that is more useful than contact area for discriminating specific
and non-specific contacts in protein crystals.
[Back to top] Complementary Peptide Epitopes and Anti-Idiotypic
Antibodies in Autoimmunity
Maria
Sakarellos-Daitsiotis, Manh-Thong Cung, Constantinos Sakarellos, Zouhair El
Hilali, Aggeliki Kosmopoulou and Chryssa Voitharou
Application of
complementary B and T cell epitopes in inducing anti-idiotypic and
anticlonotypic antibodies capable of regulating or suppressing the autoimmune
responses in experimental autoimmune myasthenia gravis (EAMG), allergic
neuritis (EAN) and allergic encephalomyelitis (EAE) has been the stimulus of
many research efforts. Studies on the idiotypic/anti-idiotypic network of anti-La/SSB
positive sera from patients with Sjogren’s Syndrome (SS) and Systemic Lupus
Erythematosus (SLE) and on animals immunized with the complementary epitopes
are presented.
[Back to top] Amyloid-b as a “Difficult Sequence” in Solid Phase Peptide Synthesis
Anna
K. Tickler, Andrew B. Clippingdale and John D. Wade
The phenomenon of
“difficult sequence” has long frustrated chemists in their efforts to assemble
peptides that contain such sequences by solid phase synthesis methods. A
variety of remedial measures are available to minimize or even abolish the
negative impact of these sequences during synthesis. These include the use of
elevated temperatures and stronger acylating reagents. Amyloid-b, a fragment of the amyloid precursor
protein, contains 40-43 residues and possesses a C-terminal sequence that is
particularly resistant to ready solid phase synthesis making it a “difficult
sequence” peptide. This review focuses on approaches to successfully assemble
the peptide by both Boc- and Fmoc solid phase synthesis.
[Back to top] Influenza A Hemagglutinin C-Terminal Anchoring Peptide:
Identification and Mass Spectrometric Study
Larisa
V. Kordyukova, Aleksander L. Ksenofontov, Marina V. Serebryakova, Tatyana V.
Ovchinnikova, Natalija V. Fedorova, Valeria T. Ivanova and Ludmila A. Baratova
MALDI-TOF MS and
N-terminal amino acid sequencing allowed us to identify several fragments of
the C-terminal peptide of Influenza A hemagglutinin (HA) containing
transmembrane domains (TMD). These fragments were detected in the organic phase
of chloroform-methanol extracts from bromelain-treated virus particles.
Heterogeneous fatty acylation of the C-terminus was revealed. Tritium
bombardment technique might open an opportunity for 3D structural investigation
of the HA TMD in situ.
[Back to top] Crystallization and Preliminary X-Ray Analysis of Complexes
of Porcine Pancreatic Elastase with Two Natural Inhibitors
Kai
Hilpert, Helga Wessner, Christa Scholz, Patrick Scheerer, Rudolf Volkmer-Engert
and Norbert Krauß
Two different
natural protease inhibitors, the squash inhibitor MCEI III and the third domain
of turkey ovomucoid inhibitor OMTKY3, were crystallized in complexes with porcine
pancreatic elastase (PPE). About 700 conditions were screened altogether.
Crystals of the complex between MCEI III and PPE were grown in citrate buffer
with and without ammonium acetate. X-ray diffraction data were collected to 1.9
Å resolution at room temperature using synchrotron radiation. The crystals
belong to space group P21, with unit-cell parameters a = 49.17, b =
44.59, c = 67.08 Å, b = 110.97°. Crystals of
the OMTKY3/PPE complex were obtained in the presence of ammonium sulfate, MES
buffer and polyethylene glycol monomethylether (PEG). These crystals of this
complex diffracted to 2.1 Å resolution and belongs to space group I222, with
unit-cell parameters a = 84.58, b = 84.61, c = 89.92 Å and diffracted to 2.2 Å
resolution. The diffraction data were collected using a conventional rotating
anode X-ray generator at room temperature. In both cases the presence of
inhibitor in the crystals was confirmed by crystallography.
[Back to top] Crystallization and Preliminary X-Ray Analysis of Plant
Class I Chitinase from Rice
Yuichiro
Kezuka, Keiji Kitazaki, Yoshikane Itoh, Jun Watanabe, Osamu Takaha, Takeshi
Watanabe, Yoko Nishizawa and Takamasa Nonaka
We report here on
crystallization and preliminary X-ray analysis of plant class I chitinase from
rice (OsChia1b). Similar single crystals of full-length OsChia1b were obtained
under two independent conditions. The crystals grown under these conditions
diffracted up to 2.1 and 2.5 Å resolution, respectively, at a synchrotron beamline,
and were found to belong to the tetragonal space group P43212.