INITIATION SITES OF PROTEIN-FOLDING BY NMR ANALYSIS

Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possib le conformations along fast routes for folding. Nuclear magnetic reson ance experiments based on the nuclear Overhauser effect (NOE) detect h ydrogen atoms close in space and provide information about local struc ture, Here we present an NMR procedure that detects almost all sequent ial NOEs between amide hydrogen atoms (H-N-H-N NOE), including those i n random coil regions in a protein, barnase, in urea solutions, A semi -quantitative analysis of these H-N-H-N NOEs identified partly structu red regions that are in remarkable agreement with those found to form early on the reaction pathway, Our results strongly suggest that the f olding of barnase initiates at the first helix and the beta-turn betwe en the third and the fourth strands. This strategy of defining residua l structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be gen erally applicable.