Protein & Peptide Letters, Volume 10, No. 6, 2003
Solid-Phase
Synthesis of Structurally Diverse Scaffolded Peptides for the Mimicry of
Discontinuous Protein Binding Sites Pp.531-539
Raimo
Franke, Christian Doll, Victor Wray and Jutta Eichler
Structural
Investigation of Proapoptotic Peptide by CD and NMR Spectroscopy Pp.541-549
Emy
Pulsinelli, Francesca Vasile, Laura Vergani, Silvio Parodi and Claudio Nicolini
Crystal
Structure of Hemoglobin from the Maned Wolf (Chrysocyon brachyurus) Using
Synchrotron Radiation
Pp.551-559
Valmir
Fadel, Fernanda Canduri, Johnny R. Olivieri, Andre L. S. Smarra, Marcio F.
Colombo, Gustavo O. Bonilla-Rodriguez
and Walter F. de Azevedo Jr.
Kinetics
and Thermodynamics of the Native and Mutated Extracellular Endoglucanases from
Cellulomonas Biazotea
Pp.561-568
M.
I. Rajoka, Yasmin Ashraf, Hamid Rashid and A.M. Khalid
Analysis
of Fibril Formation of Amyloid-b- Protein by Stretched Exponential Function Pp.569-574
Ken-ichi
Shinozaki, Takeo Konakahara, Hiroaki Okuno
and Masato Kodaka
Salting-In
Effects Offset MgCL2-Induced Refolding of Nucleoside Diphosphate Kinase Pp.575-580
Matsuiro
Ishibashi, Tsutomu Arakawa and Masao
Tokunaga
Cloning
and Expression of a New Rat Procarboxypeptidase B Gene in Escherichia Coli and
Purification of Recombination
LI
Su-Xia, ZHANG Yu-Jian, TIAN Li-Ping, YUAN Qin-Sheng and GONG Yi
A
Non-Intuitive Design of a Cyclic Decapeptide Library with Unique Backbone
Structural Features
Pp.591-597
P.K.C.Paul
The
Site-Directed Mutagenesis of Gastrodia Anti-Fungal Protein Mannose-Binding
Sites and Its Expression in Escherichia Coli Pp.599-606
Peng
Wang, Yiqin Wang, Qila Sa, Wenbin Li and Yongru Sun
Seed
Lectin from Pisum Arvense: Isolation, Biochemical Characterization and Amino
Acid Sequence Pp.607-617
Benildo
S. Cavada, Luzia I. M.M. da Silva, Marcio V. Ramos, Francisco R. Galvani,
Thalles B. Grangeiro, Katia B. Leite, Ana Maria S. Assreuy, Joao B.
Cajazeiras and Juan J. Calvete
Immobilization
of Lipases and Assay in Continuous Fixed Bed Reactor Pp.619-628
Leonice dos Reis-Costa, Andreimar M. Soares, Suzelei C. Franca, Henrique C. Trevisan, Timothy John C. Roberts
[Back to top] Solid-Phase Synthesis of
Structurally Diverse Scaffolded Peptides for the Mimicry of Discontinuous
Protein Binding Sites
Raimo
Franke, Christian Doll, Victor Wray and Jutta Eichler
Scaffolded
peptides, in which fragments of the sequence are presented through a molecular
scaffold in a discontinuous and nonlinear fashion, are promising candidates for
the mimicry of discontinuous protein binding sites. Twelve scaffold molecules
based on cyclic peptides with ring sizes ranging from 13 to 30 were generated.
Up to three different peptide fragments were attached to the scaffolds in a
site-selective manner, yielding scaffolded peptides in excellent purities, as
documented by MS, HPLC, and 2D 1H NMR spectroscopy data.
[Back to top]
Structural Investigation
of Proapoptotic Peptide by CD and NMR Spectroscopy
Emy Pulsinelli, Francesca Vasile, Laura Vergani, Silvio Parodi and Claudio Nicolini
We have performed
a systematic investigation of the structural features of the peptides Int (a
sequence able to cross cell membranes) and Int-H1(S6A,F8A) (which shows
interesting antitumoral properties). After screening in aqueous solution at
different ionic strength and pH values, we analyzed the structures of the
peptides in different water/trifluoroethanol mixtures by Circular Dichroism and
Nuclear Magnetic Resonance techniques.
[Back to top]
Crystal Structure of
Hemoglobin from the Maned Wolf (Chrysocyon brachyurus) Using Synchrotron
Radiation
Valmir
Fadel, Fernanda Canduri, Johnny R. Olivieri, Andre L. S. Smarra, Marcio F.
Colombo, Gustavo O. Bonilla-Rodriguez
and Walter F. de Azevedo Jr.
Crystal structure
of hemoglobin isolated from the Brazilian maned wolf (Chrysocyon brachyurus)
was determined using standard molecular replacement technique and refined using
maximumlikelihood and simulated annealing protocols to 1.87Å resolution.
Structural and functional comparisons between hemoglobins from the Chrysocyon
brachyurus and Homo sapiens are discussed, in order to provide further insights
in the comparative biochemistry of vertebrate hemoglobins.
[Back to top] Kinetics and Thermodynamics of the Native and Mutated
Extracellular Endoglucanases from Cellulomonas Biazotea
M.
I. Rajoka, Yasmin Ashraf, Hamid Rashid and A.M. Khalid
The mutation had
dramatic effect on the kinetic and thermodynamic parameters inferring
thermostability of endo-glucanase from Cellulomonas biazotea mutant 51 SMr .The
denaturation activation energies of native and mutated enzymes were 73.3 and
68.8 kJ/mol respectively. They showed compensation effect at 55 °C. Both
enthalpy and entropy values of irreversible thermal inactivation for mutated
enzyme were decreased suggesting that the mutation partly stabilized the
enzyme.
[Back
to top] Analysis of Fibril Formation of
Amyloid-b- Protein by Stretched Exponential Function
Ken-ichi
Shinozaki, Takeo Konakahara, Hiroaki Okuno
and Masato Kodaka
Kinetic behavior
of aggregation of amyloid-b-protein (Ab) is represented by a stretched
exponential function, F=A{1-exp(-Btn)}. Differences in temperature-dependence
of A, B and n are studied for Ab 1-40 and Ab 1-42. As the temperature is
lowered, parameter A is increased, parameter B is decreased and parameter n is
increased in Ab 1-40, while these parameters are less sensitive to temperature
in a more hydrophobic protein Ab 1-42. ln B is a linear function of n, which is
shown by ln B = -6.34n + 3.69.
[Back to top] Salting-In Effects Offset MgCl2-Induced Refolding of Nucleoside Diphosphate Kinase
Matsuiro
Ishibashi, Tsutomu Arakawa and Masao
Tokunaga
Previously we
reported that halobacterial nucleoside diphosphate kinase can be refolded in
the presence of concentrated trimethylamine N-oxide (TMAO) as well as NaCl,
indicating that enhancement of compact structure formation by TMAO is
sufficient for folding. Here we showed that the refolding effect of MgCl2 is
maximal at 1 M and declines to zero at 2 M, indicating that charge shielding
effect of MgCl2 is offset by its salting-in effect.
[Back to top] Cloning and Expression of a New
Rat Procarboxypeptidase B Gene in Escherichia Coli and Purification of
Recombination
LI
Su-Xia, ZHANG Yu-Jian, TIAN Li-Ping, YUAN Qin-Sheng and GONG Yi
A new coding
sequence of the procarboxypeptidase B gene was obtained from SD rat fresh
pancreas by RT-PCR and highly expressed in Escherichia coli in inclusion
bodies. The folded procarboxypeptidase B was subjected to trypsin enzymatic
cleavage to produce active carboxypeptidase B, subsequently, carboxypeptidase B
was effectively purified with anion exchange chromatography DEAE-FF and
hydrophobic interaction chromatography Octyl FF, as a result, 40 mg
carboxypeptidase B per litre cell culture with specific activity 7.42 u/mg was
achieved. Further research showed that the obtained recombinant carboxypeptidase
B could substitute carboxypeptidase B isolated from pancreas.
[Back to top] A Non-Intuitive Design of a
Cyclic Decapeptide Library with Unique Backbone Structural Features
P.K.C.Paul
An analysis of
hydrogen bonding patterns of cyclic decapeptide (CDP) b-sheet structures has
resulted in a 'non-intuitive' design of cyclic decapeptides wherein their b-turns and residue positions can be fixed by choosing 2 of the 10 residues,
i.e. positions i and i+4, to be Prolines or N-substituted residues. This
sequence relationship between the two Pro or N-substituted residues is shown to
uniquely define the conformation of the CDP. Furthermore, this design of the 2 b-turn,
b-sheet CDP structure is expected to be characterised by residues
disposed in an exclusive fashion in which four residues are on one side of the
ring, two on the other and the four corner residues in the b-turn are in the
plane of the ring. This opens up the possibility of fine-tuning the four
residues facing one way and /or the two residues facing the other way such that
a library containing a myriad of chemically diverse systems could be obtained.
The design process along with the molecular modelling of specific CDP’s and the
building of a CDP library are discussed in detail.
[Back to top] The Site-Directed Mutagenesis of
Gastrodia Anti-Fungal Protein Mannose-Binding Sites and Its Expression in
Escherichia Coli
Peng
Wang, Yiqin Wang, Qila Sa, Wenbin Li and Yongru Sun
Gastrodia anti-fungal
protein (GAFP) displays strong inhibitory activity against certain fungal
pathogens. Five GAFP analogues with different mutations at mannose-binding
sites and the wild-type one were expressed and purified in Escherichia coli.
The inhibitory analysis of the purified various GAFPs against the growth of
Trichoderma viride indicates that single amino acid mutated-type GAFPs have
inhibitory activity, but its activity is much less than the wild-type one. The
double and triplicate amino acids mutated GAFPs have very low inhibitory
activity. For the first time it was proved that GAFP mannosebinding sites play
key role in anti-fungi process.
[Back to top] Seed
Lectin from Pisum Arvense: Isolation, Biochemical Characterization and Amino
Acid Sequence
Benildo
S. Cavada, Luzia I. M.M. da Silva, Marcio V. Ramos, Francisco R. Galvani,
Thalles B. Grangeiro, Katia B. Leite, Ana Maria S. Assreuy, Joao B.
Cajazeiras and Juan J. Calvete
A glucose/mannose
lectin was purified by affinity chromatography from Pisum arvense seeds (PAL)
and the 50 kDa molecular mass in solution determined by size exclusion
chromatography. SDS-PAGE and electrospray ionization mass spectrometry showed
two distinct polypeptide chains: a (Mr. 5,591 Da) and b
(19,986 Da). The lectin
was extensively characterized in terms of its biochemical and biological
aspects. The amino acid sequence was established by Edman degradation of
overlapping peptides. PAL in solution behaves as a dimer and has its monomeric
structure formed by two distinct polypeptide chains named alpha (Mr. 5,591 Da)
and beta (19,986 Da) by Electrospray ionization (ESI) mass spectrometry. PAL
possesses identical amino acid sequences to that of pea seed lectin but
undoubtedly does not exhibit sequence heterogeneity. It is discussed that P.
arvense should be considered as a synonym of P. sativum. Furthermore, like pea
lectin, PAL discriminates biantennary fucosylated glycan, determined by surface
plasmon resonance.
[Back to top] Immobilization of Lipases and
Assay in Continuous Fixed Bed Reactor
Leonice
dos Reis-Costa, Andreimar M. Soares, Suzelei C. França, Henrique C. Trevisan,
Timothy John C. Roberts
Lipases are
versatile enzymes regarding the range of reactions they catalyse and substrates
on which they act. They are as well important as catalyst in organic synthesis.
Their immobilization on appropriate supports confer them greater stability
besides the possibility of operating in continuous reactors. In order to
explore these abilities, the reactions involving hydrolysis of p-nitrophenyl
acetate (PNPA) and transesterification of PNPA with n-butanol were chosen.
Lipases from two different sources were assayed, namely: microbial (Candida
rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type II). Two
immobilization methods were also used, namely: 1) adsorption, using as support
the following silica derivatives (150-300mm e 450m): phenyl, epoxy, amino and
without derivation, and 2) covalent binding, using glutaraldehyde as binding
agent and silica amino as support. This later method led to better results.
Hydrolytic activity was 6.1 U/gsupport for CRL and 0.97U/gsupport for PPL, and
of transesterification, 2,8U/gsupport for CRL and 1,9U/gsupport for PPL.
Stability of the immobilized enzyme as a function of temperature was evaluated
for CRL at 40°C and 50°C and for PPL at 32°C and 40°C. The assays were
initially carried out batchwise, both for soluble and immobilized enzymes,
aiming to the obtention of parameters for the continuos reactor. Lipases
immobilized by covalent binding were used in the assays of operacional
stability in continuos reactors. For PPL in aqueous medium, at 32°C, and CRL in
organic medium at 40°C, both operating continuously, no significant loss of activity
was detected along the analysis period of 17 days. In the case of CRL in
aqueous medium at 40°C there was a loss of activity around 40% after 18 days.
For PPL in organic medium at 40°C the loss was 33% after 20 days. Compairing
both sources with each other, very different results were obtained. Higher
activitiy was found for CRL, both for hydrolysis and for transesterification
reactions, with higher stability in organic medium. PPL showed lower activity
as well as higher stability in aqueous medium. The immobilization method by
covalent binding showed to be the most appropriate. Immobilized lipases are
therefore relatively stable both in aqueous and organic medium.