Protein and Peptide Letters - Volume 10, Issue 1, 2003

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.

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.

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.

An α-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 α-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.

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.

From the temperature dependence of fluorescence quenching kinetics, a novel temperaturesensitive 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.

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.

The GFRα1 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 GFRα1 and its mutants were evaluated in PC12 cells. The results suggest that the central domain of GFRα1 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 GFRα1.

The conformational stability and the folding process of α, β and Ψ 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 α and β are ΔG = 15.91 ± 0.28 kcal / mol, DG = 15.54 ± 2.39 kcal / mol. respectively, and Ψ trypsin is ΔG = 16.10 ± 2.51 kcal / mol. The transition curves for α, β and Ψforms suggest a molten globule state.

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

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.

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 (ε1M) and percent (ε1%) 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 (ΔGD H20) corresponding to zero guanidine concentration and the guanidine concentrations (Δ1 / 2) able to convert 50% of the molecules from the native to the unfolded state were determined. The values of ΔGD 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 ΔGD H20 and Δ1 / 2 showed that BthTX-I is the least stable among the four myotoxins assayed, with a Δ1 / 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 ΔGN-U of 0.22 and 0.41 kcal / mole, respectively. Sigmoidal curves showed welldefined two-stage transitions for both proteins.Abbreviation Used: PLA2, phospholipase A2; BthTX-I, Bothrops jararacussu bothropstoxin I, PrTX-I, Bothrops pirajai piratoxin I, MjTX-I and -II, Bothrops moojeni myotoxins I and II; GuHCl, guanidine hydrochloride; ATEE, N-acetyl-l-tyrosine ethyl ester; ATrEE, N-acetyl-l-tryptophan ethyl ester, RNAse, bovine ribonuclease A, RC-RNAse, reduced and carboxymethylated RNAse.