Protein & Peptide Letters, Volume 11, No. 2, 2004
Contents
Splicing
Of Unnatural Amino Acids Into Proteins: A Peptide Model Study Pp.107-114
Shoufa
Han and Ronald E. Viola
Conformation
Of The Cecropin-Melittin Hybrid, Cecropin A(1-8)-Melittin (1-18) Antibacterial
Peptide Pp.115-124
Savita
Tauro, Evans Coutinho and Sudha Srivastava
Electrostatic
And Hydrophobic Interactions Play A Major Role In The Stability And Refolding
Of Halophilic Proteins.
Pp.125-132
Tsutomu
Arakawa and Masao Tokunaga
Urea-Induced
Denaturation Of b-Trypsin: An Evidence For A Molten Globule State Pp.133-140
Differential
Desensitization Of Somatostatin Receptor Subtypes In Att-20 Cells Pp.141-147
Fredric
C. Mazza and Allan D. Blake
Calcineurin
Hydrolysis Of Para-Nitrophenyl Phosphorothioate Pp.149-155
Donna
J. Spannaus-Martin and Bruce L. Martin
Comparative
Study Of Interaction Of Two Type II Ribosome-Inactivating Proteins And Their
A-Chains With Model Membrane
Pp.157-163
Hong
Xu, Bao-Zhong Wang and Wang-Yi Liu
Overexpression,
Purification And Characterization Of Recombinant Salmon Calcitonin, A
Therapeutic Protein, In Streptomyces Avermitilis Pp.165-173
Chiranjib
Chakraborty, Shyam Nandi and S. Sinha
The
C-Terminus Of CamkII Is Truncated When Expressed In E. Coli Pp.175-179
M.
Praseeda, Mary K. Beena, Sarah John Asha and R.V. Omkumar
a-Amylase
Inhibitors From Ficus Sp. Seeds And Their Activities Towards Coleoptera Insect
Pests Pp.181-187
Ingrid
W.L. Bezerra, Fabiano M. Teixeira, Adeliana S. Oliveira, Carina L. Araujo, Edda
L. Leite, Karla C.S. Queiroz and Mauricio P. Sales
Protein
Extraction and Comparison of Stain Protocols For Analysis of Two-Dimensional
Electrophoresis Gels
Pp.189-194
Joana
Silva , Ana Sofia Carvalho , Rui Vitorino , Pedro Domingues , Paula Teixeira
and Paul Gibbs
Vatairea
Macrocarpa Lectin Induces Paw Edema With Leukocyte Infiltration. Pp.195-200
Nylane
M.N. Alencar, Ana M.S. Assreuy, David N. Criddle, Emmanuel P. Souza, Pedro M.G.
Soares,Alexandre Havt, Karoline S. Aragao, Daniel P. Bezerra, Ronaldo A.
Ribeiro and Benildo S. Cavada
Crystallization
and Preliminary X-Ray Crystallographic Analysis of Plasmodium Falciparum
S-Adenosyl-L-Homocysteine Hydrolase Pp.201-205
Nobutada
Tanaka, Yoshio Kusakabe, Katsura Shiraiwa, Yasumitsu Sakamoto, Masayuki
Nakanishi, Yukio Kitade and Kazuo T. Nakamura
Abstracts
[Back to top] Splicing
of Unnatural Amino Acids Into Proteins: A Peptide Model Study
Shoufa
Han and Ronald E. Viola
S-Ethyl
2-azidohexanethioate (N3-Hex-SEt), an unnatural amino acid analog of
leucine, is coupled with L-cysteine ethyl ester (NH2-Cys-OEt) to
obtain N3-Hex-Cys-OEt by native chemical ligation. Coupling of this
dipeptide with N-t-butoxycarbonyl-2-diphenylphosphinoethanethioglycinate
produces the tripeptide, t-Boc-Gly-Hex-Cys-OEt, in high yield. These reactions
suggest an approach for the incorporation of unnatural amino acids into
proteins by successive native chemical ligation and Staudinger ligation.
[Back to top] Conformation
of the Cecropin-Melittin Hybrid, Cecropin A(1-8)-Melittin (1-18) Antibacterial
Peptide
Cecropin A
(1-8)-Melittin (1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal
activity. The primary sequence of the peptide is as follows:
KWKLPKKIGIGAVLKVLTTGLPALIS-NH2. 1H and 13C 2D
NMR techniques were used to deduce the conformational parameters of chemical
shift, 3JNHa
coupling constants, temperature coefficients of NH chemical shifts and the
pattern of intra and inter-residue nOe's. NMR studies were carried out in water
(pH 6.0) and hexafluoroacetone (HFA). The peptide was found in a b-pleated structure in water, and in HFA it
adopts a right-handed a-helix conformation.
Solution structures generated using restrained molecular dynamics simulations
were refined by Mardigras to R factors ranging from 0.5 to 0.6.
[Back to top] Electrostatic and Hydrophobic
Interactions Play a Major Role in The Stability and Refolding of Halophilic
Proteins.
Tsutomu Arakawa and Masao Tokunaga
In general,
halophilic proteins are stable only in the presence of salts at high
concentrations. Not only is high salt concentration important for structural
stability of halophilic proteins, but also refolding of a denatured halophilic
protein requires high salt concentration. This review summarizes the importance
of electrostatic charge shielding and hydrophobic interactions in the stability
and refolding of halophilic proteins.
[Back to top] Urea-Induced Denaturation of b-Trypsin: An Evidence For A
Molten Globule State
Maria
Helena Nasser Brumano and Maria Goreti de Almeida Oliveira
The denaturation
of b-trypsin induced
by urea was investigated by fluorescence and circular dichroism. A transient
denatured state was found at 2 M urea in both intrinsic fluorescence spectrum
and bis-(8-anilino-1-naphtalene sulfonate) (bis-ANS) binding. In addition, the
absence of tertiary contacts and presence of secondary structure for this
state, are consistent with an intermediate equilibrium state having features of
molten globule
[Back to top] Differential Desensitization of Somatostatin Receptor Subtypes
In Att-20 Cells
Fredric
C. Mazza and Allan D. Blake
Nonpeptidyl
agonists for the somatostatin (SRIF) receptor family have been developed. We
have studied the desensitization effects for two of these agonists upon SRIF
receptor function in AtT-20 cells, a neuroendocrine tumor cell, which
endogenously expresses two distinct SRIF receptor, subtypes. We observe that
SRIF and the nonpeptidyl, subtype selective agonists, differentially regulate
SRIF receptor subtypes in the AtT-20 cell.
[Back to top] Calcineurin Hydrolysis of
Para-Nitrophenyl Phosphorothioate
Donna
J. Spannaus-Martin and Bruce L. Martin
para-Nitrophenyl
phosphorothioate (pNPT) was hydrolyzed by calcineurin at initial rates slightly,
but comparable to rates for para-nitrophenyl phosphate (pNPP). Kinetic
characterization yielded higher estimates for both Km and Vmax
compared to pNPP. Metal ion activation of phosphorothioate hydrolysis was more
promiscuous. Unlike the hydrolysis of with pNPP, Ca2+, Mg2+,
and Ba2+ activated calcineurin as well as Mn2+.
[Back to top] Comparative Study of Interaction
of Two Type II Ribosome-Inactivating Proteins and Their A-Chains With Model
Membrane
Hong
Xu, Bao-Zhong Wang and Wang-Yi Liu
Both cinnamomin
and ricin are type II ribosome-inactivating proteins. Cinnamomin is less
cytotoxic compared with ricin. In order to clarify the mechanism of their
different cytotoxicities, the interaction of cinnamomin and its A-chain with
model membrane was investigated and compared with that of ricin and its
A-chain. It was revealed that cinnamomin is less effective than ricin in
interacting with model membrane. Cinnamomin A-chain interacts with model
membrane much less violently than ricin A-chain. The differences in the
interaction of cinnamomin, ricin or their A-chains with model membrane might at
least in part indicate the different cytotoxicity between cinnamomin and ricin.
[Back to top] Overexpression, Purification and
Characterization of Recombinant Salmon Calcitonin, A Therapeutic Protein, In
Streptomyces Avermitilis
Chiranjib
Chakraborty, Shyam Nandi and S. Sinha
Calcitonin (CT) is
a peptide hormone produced by the parafollicular cells of the thyroid gland in
mammals and by the ultimobranchial gland of birds and fish. Salmon calcitonin
(sCT), which is more potent and longer lasting than human CT, has been used
widely for the treatment of osteoporosis, paget's disease, hypercalcemic shock
and chronic pain in terminal cancer patients. sCT is one of the many bioactive
peptides that require C-terminal amidation for full biological activity. In
this study we describe the over-expression and over-production of C-terminal
amidated sCT in recombinant Streptomyces avermitilis. With this approach the
utilization of expensive peptide synthesis can be circumvented.
[Back to top] The C-Terminus of CamkII is
Truncated When Expressed in E. Coli
M.
Praseeda, Mary K. Beena, Sarah John Asha and R.V. Omkumar
The neuronal
enzyme Calcium/calmodulin dependent protein kinase type II (CaMKII) is a key
molecule in biochemical events necessary for learning and memory. The a-subunit of CaMKII expressed in E. coli as
well as in insect cells shows similar catalytic behavior [Praseeda, M.,
Pradeep, K. K., Krupa, A., Sri Krishna, S., Leena, S., Rajeev Kumar, R., John
Cheriyan, Mayadevi, M., Srinivasan, N., and Omkumar, R. V. (2003) Biochem. J.
In Press]. The association domain of the enzyme has been crystallized in its
native multimeric form after expression in E. coli [Hoelz, A., Nairn, A. C. and
Kuriyan, J. (2003) Molecular Cell 11, 1241]. However a major truncation product
accompanies the full-length protein when expressed in E. coli. We show by
epitope labeling and immunoblotting that the truncation occurs at the
C-terminal half of the protein so that the N-terminal catalytic domain is
complete in the truncated product. This supports the use of the preparation of a-CaMKII expressed in E. coli for studies on
functions of the catalytic site. Our data will also be helpful in designing
modified prokaryotic expression systems for CaMKII devoid of the trun-cation
product, which are easier to use compared to the insect cell system.
[Back to top] a-Amylase Inhibitors from Ficus
Sp. Seeds and Their Activities Towards Coleoptera Insect Pests
Ingrid
W.L. Bezerra, Fabiano M. Teixeira, Adeliana S. Oliveira, Carina L. Araujo, Edda
L. Leite, Karla C.S. Queiroz and Mauricio P. Sales
a-Amylase Inhibitors were isolated from Ficus
sp. (Gameleira) seeds by acetone fractionation and Sephadex G-50. Two
inhibitors (a-PPAI and a-ZSAI)
were tested against a-amylases from
coleopteran larvae. a-PPAI was active to a-amylases of Callosobruchus maculatus (52%)
and Zabrotes subfasciatus (53%). a-ZSAI was
strongly active to Z. subfasciatus (100%) of and Mimosestes mimosae (98%). The a-ZSAI is a glycoprotein of ~50kDa with an IC50
value of 0.074 µg µl-1.
[Back to top] Protein Extraction and Comparison
of Stain Protocols For Analysis of Two-Dimensional Electrophoresis Gels
Joana
Silva , Ana Sofia Carvalho , Rui Vitorino , Pedro Domingues , Paula Teixeira
and Paul Gibbs
Protein extraction
and the proteome of Lactobacillus delbrueckii subsp. bulgaricus were studied
using different stains. The reversible silver staining technique was shown to
be more sensitive than the irreversible silver stain. Coomassie colloidal was
demonstrated to be as sensitive as reversible silver stain; however, the
Coomassie colloidal blue solution developed a higher background and for sample
preparation was more time-consuming.
[Back to top] Vatairea Macrocarpa Lectin
Induces Paw Edema With Leukocyte Infiltration.
Nylane
M.N. Alencar, Ana M.S. Assreuy, David N. Criddle, Emmanuel P. Souza, Pedro M.G.
Soares, Alexandre Havt, Karoline S. Aragao, Daniel P. Bezerra, Ronaldo A.
Ribeiro and Benildo S. Cavada
A lectin from Vatairea
macrocarpa (Vmac) seeds was investigated in a model of paw edema in rats and
the possible involvement of leukocytes. Vmac (200 and 400µg/paw, s.c.) induced
a significant time- and dose-dependent paw edema, with leukocyte infiltration,
which was drastically reduced in leukopaenic animals. These data suggest a
pro-inflammatory effect for this lectin that is dependent on the presence of
leukocytes.
[Back to top] Crystallization and Preliminary
X-Ray Crystallographic Analysis of Plasmodium Falciparum
S-Adenosyl-L-Homocysteine Hydrolase
Nobutada
Tanaka, Yoshio Kusakabe, Katsura Shiraiwa, Yasumitsu Sakamoto, Masayuki
Nakanishi, Yukio Kitade and Kazuo T. Nakamura
S-adenosyl-L-homocysteine
hydrolase from a malaria parasite Plasmodium falciparum (PfSAHH) has been
crystallized by the vapor diffusion method. The crystals belong to an
orthorhombic space group P212121 with the cell
dimensions of a = 76.66 Å, b = 86.31 Å, and c = 335.6 Å. There are four
subunits (one tetramer) per asymmetric unit. X-ray diffraction data have been
collected up to 2.8 Å resolution. Self-rotation function studies suggest that
the tetrameric PfSAHH molecule has the 222 point group symmetry.