DETECTION OF RESIDUE CONTACTS IN A PROTEIN-FOLDING INTERMEDIATE

Protein folding can be described in terms of the development of specif ic contacts between residues as a highly disordered polypeptide chain converts into the native state. Here we describe an NMR based strategy designed to detect such contacts by observation of nuclear Overhauser effects (NOEs). Experiments with alpha-lactalbumin reveal the existen ce of extensive NOEs between aromatic and aliphatic protons in the arc hetypal molten globule formed by this protein at low pH. Analysis of t heir time development provides direct evidence for near-native compact ness of this state. Through a rapid refolding procedure the NOE intens ity can be transferred efficiently into the resolved and assigned spec trum of the native state. This demonstrates the viability of using thi s approach to map out time-averaged interactions between residues in a partially folded protein.