Analysis of Asymmetry in the Distribution of Helical Residues in Peptides by ¹H Nuclear Magnetic Resonance

Peptides that assume full or partial helical structure in aqueous solution have provided useful models for investigating the determinants of α-helical structure. Circular dichroism (CD) spectroscopy, the usual measure of helicity, affords an estimate of the mean helix content when calibrated against suitable standards. Analysis of these systems by means of ¹H NMR makes it possible to determine precisely the location and extent of helix structure in a chain. NMR criteria for identifying helical domains include the following: NOE's between adjacent NH protons and between an NH proton at position i and the Cα proton at i + 3; values of the three-bond coupling constants3J_ΑN; and the relative chemical shift of the Cα protons. Application of these criteria to members of the series of partially helical synthetic peptides, succinylTyrSerGlu₄Lys₄XXXGlu₄Lys₄NH₂, in which sets of three amino acids are inserted between blocks of glutamic acid and lysine side chains, shows that the helix is located preferentially near the N terminus in chains with the central substitution Ala₃,Leu₃, as well as in the parent species lacking any substitution. The degree of helicity rises sharply at the N terminus to a maximum near residue 8 and diminishes gradually from Glul4 to the C terminus. Application of the NMR criteria to the peptide containing Gly₃ reveals very little helical structure in this peptide. These results suggest that helix formation in short chains does not conform to an all-or-none reaction.