Cooperativity of a protein folding reaction probed at multiple chain positions by real-time 2D NMR spectroscopy

The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fast formation of a partly folded intermediate is followed by the slo w reaction to the native state, limited by a trans --> cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate was de termined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by a series of 128 very fast 2D N-15-HMQC spectra, to observe the kinetics of 66 individual backbone amide probes. We find that t he intermediate is as compact as the native protein with many native chemic al shifts. All 66 analyzed amide probes follow the rate-limiting prolyl iso merization, which indicates that this cooperative refolding reaction is ful ly synchronized. The stability of the folding intermediate was determined f rom the protection factors of 45 amide protons derived from a competition b etween refolding and H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding with many protected amides in not-yet -native regions.