Using a 13C and 15N-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 3J(C',C(γ)) and 3J(N,C(γ)) coupling constants has enabled side-chain (χ1) torsion angle populations to be probed in the denatured polypeptide chain. Analysis of the coupling constant data has allowed the relative populations of the three staggered rotamers about (χ1) to be defined for 51 residues. The amino acids can broadly be divided into five classes that show differing side-chain conformational preferences in the denatured state. These range from a strong preference for the -60°(χ1) rotamer for methionine and leucine (74-79% population) to a favouring of the +60°(χ1) rotamer for threonine (67% population). The differences in behaviour reflect the steric and electrostatic characteristics of the side-chains concerned. A close agreement is seen between the (χ1) populations calculated from the experimental coupling constant data and predictions from the statistical model for a random coil that uses the (χ1) torsion angle distributions in a data base of native protein structures. Short-range interactions therefore dominate in determining the local conformational properties of side-chains in a denatured protein. Deviations are, however, observed for many of the aromatic residues involved in hydrophobic clusters within the denatured protein. For these residues the effects of additional non-local interactions in the clusters presumably play a major role in determining the (χ1) preferences.
