Effects of salt and denaturant on structure of the amino terminal alpha-helical segment of an antibacterial peptide dermaseptin and its binding to model membranes.

The amino terminal 1-18 domain of dermaseptin s is an important determinant of its structure as well as the antibacterial activity. A thorough investigation on the structure of the 18-residue peptide (D18) and its binding to model membranes in presence of salt and denaturant guanidinium chloride has been carried out. In presence of salt, there is an increase in the fraction of peptide molecules in helical conformation. In presence of the denaturant, D18 is unordered, but addition of the structure-promoting solvent trifluoroethanol results in a transition to the helical conformation. In presence of denaturant, the peptide is unordered, but binding to lipid vesicles is not abolished. Investigation of model membrane permeabilizing ability of the peptide in solutions containing various proportions of sodium chloride and guanidinium chloride indicates that vesicle permeabilization parallels extent of binding. The peptide thus binds to lipid vesicles in an unfolded state. Since the peptide has propensity to fold into a helical conformation, lipid induced transition to a helical structure occurs, followed by membrane permeabilization as a result of pore formation.

Conformations of hydrophobic peptides in trifluoroethanol, water and in solid state: a circular dichroism and Fourier Transform Infrared study.

The conformations of peptides corresponding to KLLIALVLCFLPLAALG have been examined in trifluoroethanol (TFE), aqueous medium by circular dichroism spectroscopy and in the solid state by Fourier Transform Infra Red Spectroscopy (FTIR). The 17-residue parent peptide and peptides corresponding to shorter segments LVLCFLPLAALG and CFLPLAALG showed preference for helical conformation in TFE. Even the shorter hydrophobic peptides corresponding to KLLIA and LVL showed propensity for beta-turn conformations in TFE. However, peptides corresponding to the relatively polar segment FLPLAALG were unordered in TFE. In water, peptides that showed ordered conformation in TFE preferred beta-conformation. In solid-state, FTIR spectra indicated that the hydrophobic peptides adopt beta-structures with extensive hydrogen bonded network in the solid-state. The hydrophobic core segment thus appears to dictate the conformational propensity of the peptide.

Insight into the environment of tryptophan in a hydrophobic model peptide upon aggregation and interaction with lipid vesicles: a steady state and time resolved fluorescence study.

Fluorescence spectroscopy is extensively used to monitor binding of peptides to lipid vesicles as well as orientation in the lipid bilayer. In steady-state fluorescence, the emission characteristics of intrinsic and extrinsic fluorophores, which are sensitive to environment are monitored. Life time measurements should yield useful information about the location and flexibility of fluorophores, as these factors have a significant effect on the life times. However, studies on protein structure and dynamics indicate that interpretation of life-time data is complicated (Beechem. J.M. and Brand, L. (1985) Annu. Rev. Biochem. 54, 43-71). Hence, simple well-defined systems should help in interpretation of life time data, especially in lipid-peptide interactions. In order to examine how fluorescence characteristics of tryptophan and anthroyl group would reflect molecular details of peptide aggregation and lipid-peptide interaction, studies have been carried out on a model hydrophobic peptide and its fatty acylated derivative. Steady-state fluorescence measurements suggest that: (1) the fatty acyl chain attached to an amino acid associates with the peptide chain in aqueous environment. (2) In the lipid bilayer, the acyl chain is oriented perpendicular to the lipid bilayer surface with the peptide chain at an angle to it. Analysis of the fluorescence decay of tryptophan indicates the predominance of a very short life-time component (<1ns) in aqueous environment and lipid-vesicles. Since the preexponentials were not negative, it is unlikely that this is due to extensive deactivation process. We attribute the observation of the low life time component to predominance of one rotamer around (C alpha-C beta)bond of tryptophan in aqueous and lipid environments. Our investigations suggest that fluorescence life time data need to be complemented with steady state measurements to get an insight into details of lipid-peptide interaction.

Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes.

Indolicidin is a 13-residue broad-spectrum antibacterial peptide isolated from bovine neutrophils. The primary structure of the peptide ILPWKWPWWPWRR-amide (IL) reveals an unusually high percentage of tryptophan residues. IL and its analogues where proline residues have been replaced by alanine (ILA) and trp replaced by phe (ILF) show comparable antibacterial activities. While IL and ILA are haemolytic, ILF does not have this property. Since aromatic residues would strongly favour partitioning of the peptide into the lipid bilayer interface, the biological activities of IL and its analogues could conceivably arise due perturbation of the lipid bilayer of membranes. We have therefore investigated the interaction of IL and its analogues with lipid vesicles. Peptides IL and ILA bind to lipid vesicles composed of phosphatidylcholine and phosphatidylethanol amine: phosphat idyl glycerol: cardiolipin. The position of max and I- quenching experiments suggest that the trp residues are localized at the membrane interface and not associated with the hydrophobic core of the lipid bilayer in both the peptides. Hence, membrane permeabilization is likely to occur due to deformation of the membrane surface rather than formation of transmembrane channels by indolicidin and its analogues. Peptides ILA, IL and ILF cause the release of entrapped carboxy fluorescein from phosphatidyl choline vesicles. The peptide-lipid ratios indicate that ILF is less effective than IL and ILA in permeabilizing lipid vesicles, correlating with their haemolytic activities.

Is the role of fatty acids only to provide membrane-anchor in fatty acylated proteins?

A large number of proteins on the eukaryotic cell surface that play an important role in cellular metabolism are covalently modified with fatty acids like palmitic and myristic acids. While some of these proteins have transmembrane spanning segments, many others do not. Early hypothesis was that this co or posttranslational modification helped in membrane-association and the fatty acyl chain provided a stable membrane anchor. We have investigated the structure of peptides with these modifications and also their interaction with membranes. Our results indicate that the fatty acylated peptides, especially when the peptide segment is not hydrophobic, do not have strong affinity for membranes. The recent observations about the dynamic nature of fatty acid acylation as well as the importance of protein-protein interactions for function in fatty-acylated proteins suggest that membrane-association may involve factors other than only the fatty acid, either myristic or palmitic. Revised models depicting the possible role of fatty acids in modulating protein-protein interaction and their dynamics is presented.

Acylation of proteins: recent advances.

The biochemistry and cell biology of covalent attachment of the fatty acids palmitic and myristic to proteins has been the subject of extensive investigations during the past fifteen years. While the site of attachment of fatty acids and the primary structure of proteins around the acylation site have been extensively documented, the exact role of the fatty acids have only been speculated upon. Since fatty acids would prefer to be associated with the lipid bilayer of membranes, it has been assumed that the role of the fatty acid is to provide a stable membrane anchor. This review discusses recent reports in the area of fatty acylation which suggests roles for the fatty acid other than that of a stable membrane anchor.

Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds.

An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethylcellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was 14000. The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit on A. oryzae, B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive.