Experimental characterization of polyalanine helices in short and long contexts

Abstract

Three principal analytical methods: circular dichroism (CD), hydrogen exchange, and the t/c ratio of Ac-Hel, a helix stabilizing N-cap, are used in combination to quantify fractional helicity in order to determine the intrinsic helical propensity of an alanine residue, free from the hydrophobic packing within proteins. Constructed cross checks between these methods add rigor to propensity assignments and present the opportunity to resolve controversies regarding helical propensities and limiting ellipticities used in CD. Unlike proteins that approximate a single folded conformation, simple helical polyalanine peptides exists as a manifold of partially helical conformers. The Ac-Hel template characterizes short helical conformations that exist within larger manifolds. Using an isolation and solubilization methodology, t/c measurements of Ac-Hel alanine conjugates up to 14 residues indicate an increase in the helical propensity with length that was previously undetected for 6 residue conjugates. A constructed cross check of t/c with circular dichroism ellipticities indicate that both methods provide consistent measurements of peptide helicity. Protection factors, measured by hydrogen exchange, assign site helicities that provide incisive information about the manifold of helical conformers for a fifteen residue helical alanine region. Protection factor measurements are also used to corroborate the length dependence seen by t/c over an extended length series between 5 and 25 alanine residues. A joint analysis of the experimental CD ellipticity and the fractional helicity as determined by hydrogen exchange is tested as a cross check to calibrate CD. Long helical peptides with moderately stabilizing N-caps were previously shown to exceed a fractional helicity

(cont.) of 1.0. The conformational averaging seen for intrinsic polyalanine helices can be dramatically reduced by highly stabilizing N- and C-terminal caps. An octaalanine core, stabilized by Ac-[superscript]β aspartyl-Hel as an N-cap and β-amino alanine as a C-cap, also exceeds current literature standards for factional helicity. As a proof of principle, hydrogen exchange measures factional helicity approaching 0.95 for all eight alanine residues. A complete series of this type is proposed to establish the perresidue molar ellipticity for a infinite length helix ( [theta][sub][infinity, 222]) and its length dependence.