Homonuclear Internuclear Double Resonance Spectroscopy as a Basis for Determination of Amino Acid Conformation

Abstract

INDOR (Internuclear Double Resonance) spectroscopy is shown to be superior to conventional (spectra obtained not by sweeping, but by maintaining constant the decoupling frequency) nuclear single- or double-resonance techniques for conformational studies of amino acids and amino acid residues in the following ways: (a) INDOR spectra of amino acids are inherently simpler than conventional proton magnetic resonance spectra of amino acids, and INDOR spectra of individual amino acid residues are slightly, if at all, complicated by overlap with either solvent peaks or the transitions of nuclei in other residues. (b) For each amino acid, the side-chain and C(alpha) proton belong to a particular class of spin system characterized by unique INDOR spectra, the pattern of which aids in the proper assignment of spectral lines. (c) For an amino acid with a first-order spin system, INDOR spectra directly reveal hidden chemical shifts and coupling constants. For an amino acid with a spin system other than first-order, INDOR spectra indirectly reveal values for chemical shifts and coupling constants as follows: INDOR spectra permit construction of a topological spin energy level diagram which, in turn, allows division of the PMR spectrum of the spin system into subspectra that easily yield values for chemical shifts and coupling constants. Although we only report INDOR spectra of free amino acids or amino acid derivatives that resemble amino acid residues in polypeptides, we, in effect, demonstrate a novel method to obtain total polypeptide conformation based on INDOR spectroscopy, inasmuch as the total conformation is the sum of the individual residue conformations.