Characterisation of low free-energy excited states of folded proteins

It is demonstrated that the identity of residues accessing excited conforma tional states that ape of low free energy relative to the ground state in p roteins can be obtained from amide proton NMR chemical shift temperature de pendences displaying significant curvature. For the N-terminal domain of ph osphoglycerate kinase, hen egg-white lysozyme and BPTI, conformational hete rogeneity arises from a number of independent sources, including: structura l instability resulting from deletion of part of the protein; a minor confo rmer generated through disulphide bond iso merisation; an alternative hydro gen bond network associated with buried water molecules; alternative hydrog en bonds involving backbone amides and surface-exposed side-chain hydrogen bond accepters; and the disruption of loops, ends of secondary structural e lements and chain termini. in many of these cases, the conformational heter ogeneity at these sites has previously been identified by X-ray and/or NMR studies, but conformational heterogeneity of buried water molecules has hit herto received little attention. These multiple independent low free-energy excited states each involve a small number of residues and are shown to be within 2.5 kcal mol(-1) of the ground state. Their relationship with the p artially unfolded forms previously characterised using amide proton exchang e studies is discussed. (C) 1998 Academic Press.