Protein & Peptide Letters, Volume 10, No. 1, 2003
Novel “Three-in-one” Peptide Device for
Genetic Drug Delivery Pp.1-7
Jason
Smith, Jesse Guidry, and Pernilla Wittung-Stafshede
The Temperature Dependence of Gramicidin
Conformational States in Octanol Pp.9-17
Farah
O’Boyle and B. A. Wallace
Steady-State Cleavage Kinetics For Dengue
Virus Type 2 Ns2b-Ns3(Pro) Serine Protease with
Synthetic Peptides Pp.19-26
Rabuesak
Khumthong, Pornwarat Niyomrattanakit, Santad Chanprapaph, Chanan
Angsuthanasombat, Sakol Panyim and Gerd Katzenmeier
ph-Induced Conformational Change in an a- Helical Coiled-Coil is Controlled by His
Residues
in the Hydrophobic Core Pp.27-33
Kiyoko
Wada, Toshihisa Mizuno, Jun-ichi Oku, and Toshiki Tanaka
Inhibition of Human Napsin A Pp.35-42
Rebecca
F. Cronshaw, Vesna Schauer-Vukasinovic David J. Powell, Thomas Giller, Daniel
Bur and John Kay
Direct Observation of Structure Transition of
Donor-Acceptor Labeled Peptides: Temperature
Dependence of Fluorescence Quenching Kinetics Pp.43-51
Wei
Min and Lin Sun
Kinetic and Site-Directed Mutagenesis Studies
of Prevotella Intermedia Acid Phosphatase Pp.53-59
Xiaochi
Chen, Toshihiro Ansai, Sailen Barik, Tadamichi Takehara
A Structure-Function Analysis of Glial
Cell-Linederived Neurotrophic Factor Receptor Alpha1 Pp.61-72
Li-Mei
Wang, Zhe-Yu Chen, Qing Zhang, Wei Zhu,
Da-Fu Ding, Chang-Lin Lu and Cheng He
The Denaturation of a, b and y Bovine Trypsin at Ph 3.0: Evidence of
Intermediates Pp.73-81
N.F.
Martins, E. Ferreira, K.C.L. Torres and M.M. Santoro
N-Terminal Domain Unfolds First in the
Sequential Unfolding of Papain
Pp.83-90
Yagya
Valkya Sharma and M. V. Jagannadham
Low Content of Protein S29 In Ribosomes of
Human Lung Cancer Cell Line A549: Detected by Twodimensional
Electrophoresis Pp.91-97
Zhi-Dong
Zhou, Lei Bao, Ding-Gan Liu, Min-Qian Li, Yin-Zi Ge, Ying-Ling Huang and
Wang-Yi Liu
Spectroscopic Analysis of the Stability of
Bothrops Myotoxic Phospholipases A2 to Guanidine and Urea Denaturation Pp.99-108
Ana G. Brito, Andreimar M. Soares, Maria I. Homsi-Brandeburgo, Márcia H. Borges, José R. Giglio and Nilson Penha-Silva
[Back to top] Novel “Three-in-one” Peptide Device for
Genetic Drug Delivery
Jason
Smith, Jesse Guidry, and Pernilla Wittung-Stafshede
We here describe a
new strategy for the delivery of oligonucleotides to cells that is based on the
use of a short peptide containing three functional units: a
membrane-penetrating segment, a DNA-binding domain and a cell-localization
sequence. The designed vector binds strongly to oligonucleotides and has
membrane-perturbing abilities in vitro. This type of multi-functional device
may be a powerful tool to achieve efficient delivery of genetic drugs in vivo.
[Back to top] The Temperature Dependence of Gramicidin
Conformational States in Octanol
Farah O’Boyle and B. A. Wallace
In lipid bilayers and organic solvents, the hydrophobic polypeptide gramicidin adopts a number of different conformations, some of which are capable of conducting monovalent cations across phospholipid membranes. The equilibria between conformations have been shown to be influenced by factors such as lipid chain length, solvent, concentration and salt. In this study, the temperature dependence of the equilibrium mixture of double helical ion-free gramicidin in octanol was examined using circular dichroism spectroscopy.
[Back to top] Steady-State Cleavage Kinetics for Dengue
Virus Type 2 Ns2b-Ns3(Pro) Serine Protease with
Synthetic Peptides
Rabuesak
Khumthong, Pornwarat Niyomrattanakit, Santad Chanprapaph, Chanan
Angsuthanasombat, Sakol Panyim and Gerd Katzenmeier
The N-terminal
part of the NS3 protein from dengue virus contains a trypsin-like serine
protease responsible for processing the nonstructural region of the viral
polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a
full-length NS2B cofactor of dengue virus type 2 was examined by using
synthetic dodecamer peptide substrates encompassing native cleavage sequences
of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions.
Cleavage of the dansylated substrates was monitored by a HPLC-based assay and
kinetic parameters for Km, kcat and kcat/Km
were obtained. The data presented here show that NS2B-NS3(pro) expressed in
recombinant E. coli can be renatured to an active protease which reacts in the
absence of microsomal membranes with all 4 substrate peptides, albeit the
molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A
marked difference in cleavage efficiency was found for the NS2B/NS3 substrate
and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5
cleavage sites.
[Back to top]
pH-Induced Conformational Change In an a- Helical Coiled-Coil is Controlled by His
Residues
in the Hydrophobic Core
Kiyoko
Wada, Toshihisa Mizuno, Jun-ichi Oku, and Toshiki Tanaka
An a-helical coiled-coil structure is one of the
basic structural units in proteins. Hydrophilic residues at the hydrophobic
positions in the coiled-coil structure play important roles in structures and
functions of natural proteins. We reported here a peptide that formed a triple
stranded a-helical coiled-coil showing the pH-dependent
structural change. The peptide was designed to have two His residues at the
hydrophobic positions of the center of the coiled-coil structure. The peptide
folded into a triple stranded coiled-coil at neutral pH, while it unfolded at
acidic pH. This construct is useful to create a protein that the structure or
function is controlled by pH.
[Back to top] Inhibition of Human Napsin A
Rebecca
F. Cronshaw, Vesna Schauer-Vukasinovic David J. Powell, Thomas Giller, Daniel
Bur and John Kay
The
newly-discovered human aspartic proteinase, napsin A was not susceptible to
protein inhibitors from potato, squash or yeast but was weakly inhibited by the
17 kDa polypeptide from Ascaris lumbricoides and potently by isovaleryl and
lactoyl-pepstatins. A series of synthetic inhibitors was also investigated
which contained in the P1-P1¢ positions the dipeptide analogue statine or
its phenylalanine or cyclohexylalanine homologues and in which the residues
occupying P4-P3¢ were varied systematically. On this basis,
the active site of napsin A can be readily distinguished from other human
aspartic proteinases.
[Back to top] Direct Observation of Structure Transition of
Donor-Acceptor Labeled Peptides: Temperature
Dependence Of Fluorescence Quenching Kinetics
Wei
Min and Lin Sun
From the
temperature dependence of fluorescence quenching kinetics, a novel
temperature-sensitive structure transition was discovered in a donor-acceptor
labeled peptides. Perfect agreement of related abrupt changes in a1,t1 andt2 gives strong support for the existence of this structure transition,
which suggests it is possible to develop peptides that can be rapidly and
reversibly switched between two structure states in response to temperature
shift.
[Back to top] Kinetic and Site-Directed Mutagenesis Studies
of Prevotella Intermedia Acid Phosphatase
Xiaochi
Chen, Toshihiro Ansai, Sailen Barik, Tadamichi Takehara
Site-directed
mutagenesis was used to examine the roles of the conserved histidine, arginine
and cysteine residues in acid phosphatase from Prevotella intermedia (PiACP).
The replacement of histidine and arginine residues resulted in the elimination
of the PiACP activity while the cysteine mutants retained activity. These
results suggest that the histidine and arginine residues are essential for
catalysis.
[Back to top] A Structure-Function Analysis of Glial
Cell-Linederived Neurotrophic Factor Receptor Alpha1
Li-Mei
Wang, Zhe-Yu Chen, Qing Zhang, Wei Zhu,
Da-Fu Ding, Chang-Lin Lu and Cheng He
The GFRa1 cDNA was amplified by RT-PCR from fetal rat
hippocampus. The soluble recombinant GFRa1 and its mutants were obtained from an
Escherichia coli expression system. The biological activity of soluble GFRa1 and its mutants were evaluated in PC12
cells. The results suggest that the central domain of GFRa1 is a crucial determinant for ligand
binding. This established a solid basis for further study to find the key amino
acid mediating the binding of GDNF and GFRa1.
[Back to top] The Denaturation of a, b
and y Bovine Trypsin at ph 3.0: Evidence of
Intermediates
N.F.
Martins, E. Ferreira, K.C.L. Torres and M.M. Santoro
The conformational
stability and the folding process of a, b and y
bovine trypsin at pH 3.0 followed by circular dichroism (CD) and size exclusion
in HPLC have been analyzed as a function of urea concentration. The
thermodynamic stability for a and b are
DG = 15.91 ± 0.28 kcal/mol, DG = 15.54 ± 2.39 kcal/mol. respectively, and
y trypsin is DG = 16.10 ± 2.51 kcal/mol. The transition
curves for a, b and
y forms suggest a molten globule state.
[Back to top] N-Terminal Domain Unfolds First in the Sequential
Unfolding of Papain
Yagya
Valkya Sharma and M. V. Jagannadham
Temperature and
Guanidine hydrochloride induced unfolding transitions of papain at pH 2.0 are
biphasic implying independent and sequential unfolding of its two domains. To
determine the order of unfolding, the active site located in the interface of
the domains was labeled with an environment specific fluorescent probe
(1,8-IAEDANS). Unfolding of this complex relative to the free protein followed
by intrinsic and extrinsic fluorescence measurements suggests that the N domain
unfolds initially in the sequential unfolding of domains
[Back to top] Low Content of Protein S29 in Ribosomes of
Human Lung Cancer Cell Line A549: Detected by Twodimensional Electrophoresis
Zhi-Dong
Zhou, Lei Bao, Ding-Gan Liu, Min-Qian Li, Yin-Zi Ge, Ying-Ling Huang and
Wang-Yi Liu
This study
revealed that the content of protein S29 in ribosomes of cancer cell line A549
was distinctly low (equivalent to about 30% of that of 2BS). The conclusion was
acquired based on the ratios of spot volume of ribosomal protein S29 to that of
several other ribosomal proteins (S29/L37a, S29/L38, S29/S27 and S29/S28) in
the same gel plate. The possible biological roles of ribosomal protein S29 in
malignant transformation and translation regulation are briefly discussed.
[Back to top] Spectroscopic Analysis of the Stability of
Bothrops Myotoxic Phospholipases A2 to Guanidine and Urea Denaturation
Ana
G. Brito, Andreimar M. Soares, Maria I. Homsi-Brandeburgo, Márcia H. Borges,
José R. Giglio and Nilson Penha-Silva
Spectrophotometric
profiles representing the unfolding induced by guanidine on Bothrops moojeni
myotoxins-I (MjTX-I) and II (MjTX-II), Bothrops jararacussu bothropstoxin-I
(BthTX-I) and Bothrops pirajai piratoxin-I (PrTX-I) were obtained and compared
with those obtained with bovine ribonuclease A (RNAse) and trypsin. The molar (e1M) and percent (e1%) extinction coefficients were determined for the four myotoxins as
well as for RNAse and trypsin as reference parameters. These coefficients were
then used throughout this work. The changes in free energy (DGDH20)
corresponding to zero guanidine concentration and the guanidine concentrations
(D1/2) able to convert 50% of the molecules from the native to the
unfolded state were determined. The values of DGD H20
ranged from 4.42 (BthTX-I) to 8.02 (MjTX-I) kcal/mole, compared with 6.47 and
6.88 kcal/mole for trypsin and RNAse, respectively. The values for DGDH20 and
D1/2 showed that BthTX-I is the least stable among the four myotoxins assayed,
with a D1/2 close to that of RNAse, while MjTX-II is conformationally the most
stable. Monitoring of the unfolding of RNAse and PrTX-I by a 0 to 6 M urea
gradient PAGE revealed transitions from the native (N) to the unfolded (U)
state with DGN-U of 0.22 and 0.41 kcal/mole, respectively. Sigmoidal
curves showed well-defined two-stage transitions for both proteins.