Studies on Peptides of conformational and biological interest: chemotactic peptide analogs, acyclic cystine peptide and fragments of bacteriorhodopsin and emerimicin

Abstract

Studies on the physical and conformational properties of oligopeptides have been stimulated by their widespread occurrence, impressive range of biological functions, and wide array of structural motifs. This thesis attempts to explore the conformational requirements for biological activity in two classes of peptides: (1) chemotactic peptides, and (2) ??aminoisobutyric acid (Aib) containing peptide fragments of the antibiotics, emerimicin III and IV. It also describes studies on the conformational preferences of acyclic cystine peptides and hydrophobic fragments of the bacteriorhodopsin G?helix. This thesis is divided into 9 chapters.

Chapter I provides an introduction to polypeptide structures and a brief survey of the physical methods used in their conformational analysis.

Chapter II describes the synthesis of a few chemotactic tripeptide analogs with modifications in positions 1, 2, and 3 of the parent chemotactic peptide, For-Met-Leu-Phe-OH, and compares their ability to release lysozyme from rabbit neutrophils. It is observed that the conformationally constrained analogs containing Aib and 1-aminocyclopentanecarboxylic acid (Acc) at position 2 in the parent peptide exhibit high biological activity. Interestingly, the dimeric analog, S,S'-bis(For-Cys-Leu-Phe-OMe), is found to be significantly active.

Chapter III presents a detailed conformational analysis of a few highly biologically active chemotactic tripeptide analogs in a wide variety of solvents using ¹H NMR, ¹³C NMR, IR, and CD methods. The crystal structure and solution conformation of Boc-Met-Aib-Phe-OMe, which is related to the biologically active chemotactic peptide analog For-Met-Aib-Phe-OMe, are also described. It is observed that the stereochemically constrained analogs prefer folded ?-turn conformations in solution, suggesting that the chemotactic peptide receptor in rabbit neutrophils recognizes peptides with folded structures. A model for the rabbit neutrophil chemotactic peptide receptor topology consistent with these results has been proposed.

Chapter IV describes the influence of conformation on peptide aggregation in organic solvents. Spectroscopic studies of two chemotactic peptide analogs, For-Met-Leu-Phe-OMe and For-Met-Aib-Phe-OMe, which have sequences with markedly different tendencies to adopt folded conformations, are described. The effect of conformation on peptide aggregation in the aqueous phase, using dansylated peptides Dans-Met-Leu-Phe-OMe and Dans-Met-Aib-Phe-OMe as monitored by fluorescence spectroscopy, is also described. The concentration dependence of spectroscopic data suggests that peptide aggregation is a more facile process in an extended conformation compared to a folded peptide conformation.

Chapter V deals with the synthesis and spectroscopic studies of a few acyclic cystine derivatives, including S,S'-bis(Boc-Cys-NHMe), S,S'-bis(Boc-Cys-OMe), S,S'-bis(Boc-Cys-Leu-Ala-OMe), S,S'-bis(Boc-Ala-Cys-Leu-OMe), S,S'-bis(Boc-Ala-Leu-Cys-OMe), S,S'-bis(Boc-Ala-Gly-Cys-Leu-Phe-OMe), and S,S'-bis(Boc-Val-Gly-Cys-Leu-Ala-OMe). The spectroscopic data suggest that the cystine residue introduces a strong constraint on the peptide backbone, even in acyclic sequences, and stabilizes intramolecular antiparallel ?-sheet structures.

Chapter VI describes the synthesis and spectroscopic studies of hydrophobic peptides that form part of the G-helix of bacteriorhodopsin. The sequences examined are Boc-Gly-Ile-Val-Pro-Leu-OMe (197–201), Boc-Gly-Ala-Gly-Ile-Val-Pro-Leu-OMe (195–201), and Boc-Gly-Ala-Gly-Ile-Val-Pro-Leu-Asn-Ile-OMe (195–203). NMR data in polar hydrogen-bonding solvents (CD?SOCD?) suggest that these fragments prefer non-hydrogen bonded structures. In low-polarity solvents such as chloroform, the peptides aggregate to form ?-sheet structures, as evident from ¹H NMR and IR data. CD data suggest that these hydrophobic sequences are structurally flexible and generally assume ?-sheet structures in nonpolar solvents, whereas helical conformations appear to be populated in polar solvents.

Chapter VII gives a brief description of the synthesis of fragments of the peptide antibiotics emerimicin III and IV: Emerimicin III (IV): Ac-Phe-(Aib)?-Val-Gly-Leu-(Aib)?-Hyp-Gln-Iva-Hyp-Ala(Aib)-Phol. Their ability to function as uncouplers of oxidative phosphorylation in rat liver mitochondria is compared. The synthesis of 1–10 and 11–15 segments of the polypeptide is described. Difficulties encountered in synthesizing the polar carboxy-terminal pentapeptide Boc-Gln-Aib-Hyp-Aib-Phol are also presented. Longer fragments are effective uncouplers of oxidative phosphorylation in rat liver mitochondria, whereas shorter fragments up to the hexapeptide are inactive.

Chapter VIII summarizes NMR and IR studies in solution of fragments of emerimicin III and IV. The amino-terminal pentapeptide (Boc-Phe-(Aib)?-Val-OMe) and carboxy-terminal pentapeptide (Boc-Gln-Aib-Hyp-Aib-Phol) favor 3??-helical conformations in solution, while the central pentapeptide fragment (Boc-Gly-Leu-Aib-Aib-Hyp-OMe) adopts a single ?-turn conformation. Spectroscopic data of the amino-terminal fragments from tetrapeptide to hexapeptide are consistent with 3??-helical conformations in CDCl? and (CD?)?SO. For longer fragments (heptapeptide to decapeptide), NMR studies support 3??-helical conformations in CDCl? but suggest different conformations in (CD?)?SO. ?-helical conformations are consistent with spectral data in (CD?)?SO.

Chapter IX summarizes the major findings of this investigation.

Publications based on this work:

Conformational effects on peptide aggregation in organic solvents. Spectroscopic studies of two chemotactic peptide analogs, P. Antony Raj and P. Balaram, Biopolymers (1985), in press.

Conformations of peptides containing 1-aminocyclohexanecarboxylic acid (Acc). Crystal structure of two model peptides, R. Bardi, A.M. Piazzesi, C. Toniolo, M. Sukumar, P. Antony Raj, and P. Balaram, Int. J. Peptide Protein Res. (1985), in press.

A highly active chemotactic peptide analog incorporating the unusual residue 1-aminocyclohexanecarboxylic acid at position 2, M. Sukumar, P. Antony Raj, P. Balaram, and E.L. Becker, Biochem. Biophys. Res. Commun. 128, 339–344 (1985).

Solid-state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. ?-turn conformation of a sequence related to an active chemotactic peptide analog, R. Bardi, A.M. Piazzesi, C. Toniolo, P. Antony Raj, S. Ragothama, and P. Balaram, Int. J. Peptide Protein Res. (1985), in press.