Nuclear magnetic resonance and optical spectroscopic studies of poly l and poly d alanine

Abstract

Nuclear magnetic resonance spectroscopy is finding increasing application to studies of conformational changes in biological macromolecules and their synthetic analogues. It has been suggested that the resonance peaks due to backbone protons in helical polymers will be broadened by non-averaging of dipole-dipole interactions and that the areas of these resonance peaks will be a measure of the random coil content of the system. In agreement with others, we do not find this to be the case for poly l and poly d alanine in trifluoracetic acid (TFA)-deuterochloroform (CDCl 3) solvent systems where the optical rotatory dispersion parameters indicate a helix content in excess of 50 per cent while the n.m.r. spectra for these solutions are still fully developed. It is suggested that the peak narrowing process operating in this system is rapid exchange between random coil and helical segments. Others suggest that, although the b 0 values for the TFA-CDCl 3 solvent systems indicate 54 per cent helix content, this o.r.d. parameter is an unreliable guide to helix content and that the polypeptide is helical; furthermore that they are observing for the first time a well developed n.m.r. spectrum for a polypeptide ‘purported to be in a helical conformation’. These conclusions are not supported by the results obtained from studies of poly l and poly d alanine in dichloracetic acid (DCA)-TFA and DCA-CDCl 3 solvent systems. In these systems it is possible to obtain appreciably higher b 0 values (up to 458°) than was observed for the TFA-CDCl 3 solvent system and these increases in b 0 are accompanied by marked broadening and loss of area of the proton resonance peaks. It is suggested therefore that when the helix content is greater than 50 per cent the motions of helical segments become too slow for the peak narrowing process of exchange between helical and random coil forms to be effective. These observations support the earlier suggestions that in fully helical macromolecules the backbone protons resonance peaks will be broadened by non-averaging of dipole-dipole interactions. It is possible to correlate the shifts in the poly l alanine resonance peaks with helix content and it has been suggested that the shifts are due to exchange of the protons between two chemical environments, i.e. the helical and random coil forms. Infra-red studies show, however, that there is a specific interaction between the carboxylic acid molecule and the helical form of poly alanine and we suggest that part of the shifts observed in the resonance peaks may result from magnetic anisotropy effects of the carboxyolic acid molecules complexed with hydrogen bonded amide groups in the helical form. In agreement with others it is suggested that the mode of interaction of the carboxylic acid molecules with poly alanine is a strong hydrogen bonding interaction and not protonation as has been suggested.