Side-chain conformations in an unfolded protein: chi(1) distributions in denatured hen lysozyme determined by heteronuclear C-13, N-15 NMR spectroscopy

Using a C-13 and N-15-labelled sample, multi-dimensional heteronuclear NMR techniques have been carried out to characterise hen lysozyme denatured in 8 M urea at pH 2.0. The measurement of (3)J(C',C-gamma) and (3)J(N, C-gamma ) coupling constants has enabled side-chain chi(1) torsion angle population s to be probed in the denatured polypeptide chain. Analysis of the coupling constants data has allowed the relative populations of the three staggered rotamers about chi(1) to be defined for 51 residues. The amino acids can b roadly be divided into five classes that show differing side-chain conforma tional preferences in the denatured state. These range from a strong prefer ence for the -60 degrees chi(1) rotamer for methionine and leucine (74-79% population) to a favouring of the +60 degrees chi(1) rotamer for threonine (67% population). The differences in behaviour reflect the steric and elect rostatic characteristics of the side-chains concerned. A close agreement is seen between the chi(1) populations calculated from the experimental coupl ing constant data and predictions from the statistical model for a random c oil that uses the chi(1) torsion angle distributions in a data base of nati ve protein structures. Short-range interactions therefore dominate in deter mining the local conformational properties of side-chains in a denatured pr otein. Deviations are, however, observed for many of the aromatic residues involved in hydrophobic clusters within the denatured protein. For these re sidues the effects of additional non-local interactions in the clusters pre sumably play a major role in determining the chi(1) preferences. (C) 1999 A cademic Press.