Protein & Peptide Letters, Volume 10, No. 4, 2003
Contents
Obtaining
Site-Specific Calcium-Binding Affinities of Calmodulin Pp.331-345
Jenny
J. Yang , Amy Gawthrop and Yiming Ye
An
Antiparallel b-Sheet and a b-Turn Characterize the Structure of Antiviral HIV-1 Peptide
T140, as Revealed by 2D NMR and MD Simulations. Pp.346-360
Savita
Tauro , Evans Coutinho and Sudha
Srivastava
Mutation
of the Hydrophobic Residue on Helix a5 of the Bacillus Thuringiensis CRY 4B Affects Structural
Stability. Pp.361-368
Chartchai
Krittanai , Apichai Bourchookarn, Wanwarang Pathaichindachote and Sakol Panyim
Protein
Production by Stationary Phase Induction (SPI) Pp.369-374
A
Non-Active Site Residue, Cysteine 69, of Glutathione S-Transferase Adgstd3-3
has a role in Stability and Catalytic Function. Pp.375-385
Jeerang
Wongtrakul, Issara Sramala and Albert J. Ketterman
Proliferative
Activity of Neokyotorphinrelated Hemoglobin Fragments in Cell Cultures Pp.386-395
Olga
V. Sazonova , Elena Yu. Blishchenko, Olga A. Kalinina, Natalya S. Egorova, Andrei
Yu. Surovoy, Marina M. Philippova, Andrei A. Karelin and Vadim T. Ivanov
Inhibitory
and Promotive Effects of Polyamines on Transglutaminase-Induced Protein
Polymerization Pp.396-403
Naoko
Sato , Yosuke Ohtake, Hiroyuki Kohno, Shinya Abe and Yasuhito Ohkubo
Production
and Purification of Recombinant Human Oxytocin Overexpressed as a Hybrid
Protein in Escherichia Coli
Pp.404-411
Roman
S. Esipov , Larisa A. Chupova, Sergey V. Shvets, Dmitry V. Chuvikovsky,
Alexandr I. Gurevich, Tatyana I. Muravyova and Anatoly I. Miroshnikov
Crystallization
of the Terminal Oxygenase Component of Biphenyl Dioxygenase Derived from
Rhodococcus Sp. Strain RHA1
Pp.412-417
Venugopalan
Nagarajan , Nobuyuki Sakurai , Miho Kubota , Takamasa Nonaka , Hiroaki Nagumo ,
Hisashi Takeda , Tomoko Nishizaki , Eiji Masai , Masao Fukuda , the late Yukio
Mitsui and Toshiya Senda
Crystallization
and X-Ray Analysis of NH3- Dependent NAD+ Synthetase from
Helicobacter pylori
Pp.418-421
Gil
Bu Kang, Yun Sik Kim, Young Jun Im, Seong-Hwan Rho and Soo Hyun Eom
Crystallization
and Preliminary X-Ray Diffraction Study of Hemoglobin D from the Aldabra Giant
Tortoise, Geochelone gigantea
Pp.422-425
Takao Kuwada , Tomokazu Hasegawa , Isamu Satoh , Koichi Ishikawa and Fumio Shishikura
[Back to top] Obtaining
Site-Specific Calcium-Binding Affinities of Calmodulin
Jenny
J. Yang , Amy Gawthrop and Yiming Ye
Calmodulin (CaM)
is an EF-hand Ca(II)-binding protein involved in the regulation of many important
biological processes. To date, there is a wealth of information available
concerning studies to obtain site-specific calcium binding affinities of CaM,
and further to estimate the cooperativity of calcium binding using mutational
studies, peptide models, and proteolytic fragmentation. In this paper, we will
discuss the energetics of calcium binding and the strong relationship between
calcium binding cooperativity and conformational change. We then explain the
difficulty of studying key determinants of calcium binding affinity of CaM due
to the large change of calcium binding affinity upon mutation. Subsequently, we
will introduce “grafting” as a novel approach to obtain the site-specific metal
binding properties of calmodulin.
[Back to top]
An Antiparallel b-Sheet and a b-Turn Characterize the Structure
of Antiviral HIV-1 Peptide T140, as Revealed by 2D NMR and MD Simulations.
Savita Tauro , Evans Coutinho and Sudha Srivastava
The polyphemusins present
in the hemocytes of the horsechoe crab and their structurally modified analogs
have been shown to exhibit activity against HIV-1. Among the many variants, T22
([Tyr5,12, Lys7]-polyphemusin II), and its shorter and
more potent analog, T140 [Arg1-Arg-2-Nal-Cys-Tyr5-
Arg-Lys-D Lys-Pro-Tyr10-Arg-Cit-Cys-Arg14] (Polyphemusin
II-derived peptide), affect the HIV-cell fusion process and inhibit the T-cell
line-tropic (T-tropic) HIV-1 infection. Conformational studies of polyphemusin
II derived peptide have been carried out by 1H and 13C
2D-NMR and MD simulations in water and HFA (40%). The NMR parameters of
chemical shift, temperature coefficients of the NH chemical shifts, 3JNHa coupling constants and the pattern of nOe’s
were used to deduce the structural characteristics. Solution structures were
generated using dihedral and distance restraints by MD simulations. The
structures are characterized by a dominant family possessing an anti-parallel b-pleated sheet that is constrained by the
disulphide bridge between Cys4 and Cys13. The two strands of the b-sheet are joined by a Type II' b-turn spanning the residues Lys7-D-Lys8-Pro9-Tyr10.
This conformation is present in both water and HFA. The only difference in the
two structures is that the b-strands are more cohesive in HFA being firmly held
by H-bonds. The solution structures generated from MD simulations were refined
by MARDIGRAS to R-factors of 0.44 and 0.57 in water and HFA respectively. The
conformation deduced for T140 is very similar to that reported for T22 and is
thought to be associated with their anti HIV activity.
[Back to top]
Mutation of the
Hydrophobic Residue on Helix a5 of the Bacillus Thuringiensis CRY 4B Affects Structural
Stability.
Chartchai
Krittanai , Apichai Bourchookarn, Wanwarang Pathaichindachote and Sakol Panyim
Cry4B toxin is a
mosquito-larvicidal protein from the Bacillus thuringiensis subsp. israelensis.
We have investigated the role of two conserved hydrophobic residues of Cry4B in
structural stabilization. Substitutions of the leucine-175 and isoleucine-189
on helix a5 with valine and leucine did not affect the expression level,
solubility and proteolytic processing. Steady state analysis of an unfolding
experiment as monitored by circular dichroism and fluorescence spectroscopy
demonstrated a typical two-state transition. The determined unfolding free
energy for the L175V mutant revealed a structural destabilization of 10.49
kcal/mol relative to the wild type. However unfolding kinetic analysis gave
identical activation energy for wild type and both mutants. Our findings
suggested that a perturbation on the close packing of the hydrophobic side
chains in protein interior could lead to a significant destabilization of the
native conformation.
[Back to top] Protein Production by Stationary Phase Induction (SPI)
Young
Kee Chae , Kyoung Suk Cho, Woochun Chun, Kyunghee Lee
An alternative
method for expressing the recombinant proteins in Escherichia coli is proposed.
Unlike the ordinary induction protocol where the cells in the early- to mid-log
phase are induced for the protein production, this alternative protocol
utilizes the cells in the stationary phase. By using a glutathione
S-transferase fusion protein as an example, the protocol proposed in this
report yielded a higher amount of the desired protein than that from the
ordinary protocol. This protocol also suppressed the proteolytic cleavage of
the desired protein in the Escherichia coli cytoplasm, thus it should be particularly
useful to produce proteins that undergo unwanted cleavages.
[Back
to top] A Non-Active Site Residue,
Cysteine 69, of Glutathione S-Transferase Adgstd3-3 has a role in Stability and
Catalytic Function.
Jeerang
Wongtrakul, Issara Sramala and Albert J. Ketterman
The Cys69 residue
of an Anopheles dirus glutathione S-transferase isoform (adGSTD3-3), was
characterized to elucidate its contribution in both catalysis and structural support.
Nine mutants were generated at this position by replacing the residue with
polar, non-polar and charged residues. The polar residues changed the Vm
of the enzymes. With non-polar residues, the enzymes were unable to fold and
were expressed in the insoluble inclusion form. With charged residues, the
soluble enzyme yields were only 3% of the wild type protein. Molecular dynamics
simulation also was performed to understand the changes in the enzyme
structure. These findings are additional evidence of the importance of
structural residues that affect the enzymatic properties such as Vm,
Km and enzyme specificity.
[Back to top] Proliferative Activity of
Neokyotorphinrelated Hemoglobin Fragments in Cell Cultures
Olga
V. Sazonova , Elena Yu. Blishchenko, Olga A. Kalinina, Natalya S. Egorova,
Andrei Yu. Surovoy, Marina M. Philippova, Andrei A. Karelin and Vadim T. Ivanov
a-Hemoglobin fragments a-(133-141), a-(134-141), a-(135-141), a-(137-141),
a-(134-140), a-
(133-138), a-(134-140) and a-(137-138)
stimulate L929 tumor cell proliferation, a-(134-141)
being the most active. a-(134-141)
stimulates proliferation of M3 melanoma cells, murine embryonic fibroblasts,
primary cultures of red bone marrow and spleen cells. In L929 cells the effect
of a-(134-141) is cell density independent; in M3
cells a-(137-141) and a-(134-141) are most active at
density 10,000 cells/well (96 well plate) independently on FBS content.
[Back to top] Inhibitory and Promotive Effects
of Polyamines on Transglutaminase-Induced Protein Polymerization
Naoko
Sato , Yosuke Ohtake, Hiroyuki Kohno, Shinya Abe and Yasuhito Ohkubo
Transglutaminase
(TGase) has been reported to be involved in the regulation of cell growth. We
examined the effects of polyamines on TGase activity. The polymerization of
casein was inhibited by putrescine (PUT) and spermidine (SPD). On the other
hand, polymerization of N,N-dimethylcasein was increased by spermine (SPM) and
SPD. These results suggested polyamines played two distinct roles as inhibitor
and promoter for TGase-catalyzed protein polymerization.
[Back to top] Production and Purification of
Recombinant Human Oxytocin Overexpressed as a Hybrid Protein in Escherichia Coli
Roman
S. Esipov , Larisa A. Chupova, Sergey V. Shvets, Dmitry V. Chuvikovsky,
Alexandr I. Gurevich, Tatyana I. Muravyova and Anatoly I. Miroshnikov
The plasmid DNA
pERilox4 containing the gene of the recombinant protein, which included the
leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high
level of gene expression in E. coli was achieved. The method for purification
of the recombinant protein and its isolation in the soluble form was developed.
The conditions for digestion of the hybrid protein by trypsin and
carboxypeptidase B were matched. The effective method for transformation of
oxytocinic acid to oxytocin was worked out. The scheme suggested allowed
obtaining oxytocin in high yield.
[Back to top] Crystallization of the Terminal
Oxygenase Component of Biphenyl Dioxygenase Derived from Rhodococcus Sp. Strain
RHA1
Venugopalan
Nagarajan , Nobuyuki Sakurai , Miho Kubota , Takamasa Nonaka , Hiroaki Nagumo ,
Hisashi Takeda , Tomoko Nishizaki , Eiji Masai , Masao Fukuda , the late Yukio
Mitsui and Toshiya Senda
The terminal
oxygenase component of the biphenyl dioxygenase (BphA1A2 complex) was
over-expressed with a novel over expression system in recombinant Rhodococcus
strain and purified. The purified enzyme has been crystallized by the hanging
drop vapor diffusion method and subjected to X-ray diffraction analysis. The
crystals belong to the tetragonal system in the space group P41212
or P43212 and diffract to better than 2.2Å resolution.
[Back to top] Crystallization
and X-Ray Analysis of NH3- Dependent NAD+ Synthetase from
Helicobacter pylori
Gil
Bu Kang, Yun Sik Kim, Young Jun Im, Seong-Hwan Rho and Soo Hyun Eom
The ubiquitous NAD+
synthetase catalyzes the key step in the biosynthesis of nicotinamide adenine
dinucleotide. NH3-dependent NAD+ synthetase from Helicobacter pylori
was purified to homogeneity and crystallized using PEG 1500 as a preciptant.
The crystal diffracted up to a resolution of 2.3+ and was found to belong to
space group C2 with unit cell dimensions of a = 93.8, b = 48.3, c = 64.2 Å and a = g =
90, b = 110.0°.
[Back to top] Crystallization and Preliminary X-Ray
Diffraction Study of Hemoglobin D from the Aldabra Giant Tortoise, Geochelone
gigantea
Takao
Kuwada , Tomokazu Hasegawa , Isamu Satoh , Koichi Ishikawa and Fumio Shishikura
Hemoglobin D (Hb
D) from the Aldabra giant tortoise, Geochelone gigantea, was crystallized by
the hanging drop vapor diffusion technique with a precipitant solution
containing 10% polyethylene glycol 3350 and 50 mM HEPES-Na, pH 7.5. The Hb D
crystals of G. gigantea, which diffract to at least a 2.0 Å resolution, belong
to the monoclinic space group C2 with unit cell dimensions of a = 112.1 Å, b =
62.4 Å, c = 54.0 Å, and b = 110.3°.
One ab dimer molecule of Hb D existed in an
asymmetric unit, with a calculated value of Vm of 2.77 Å3Da-1.