Experimental studies of folding kinetics and structural dynamics of small proteins

Main points given in the above reports can be summarized as follows. Multiple unfolded forms exist for lysozyme as well as for RNase. The existence of fast- and slow-folding forms appears to be a general phenomenon; it has been confirmed for a number of globular proteins, which contain proline residues. The major slow refolding reaction of RNase A is a sequential process via structural intermediates. A rapidly formed intermediate has also been detected on the direct UF----N refolding pathway of lysozyme. The activated state for folding of lysozyme shows a conformation similar to the native protein in terms of packing of hydrophobic groups. This suggests that, in terms of compactness, the rate-limiting step occurs at a late stage of the refolding process. A protein homologous to lysozyme, alpha-lactalbumin, shows similar kinetics although alpha-lactalbumin shows an apparent equilibrium unfolding intermediate. The location of the rate-limiting step close to the native state has also been suggested for other proteins. It still remains open whether this is a general property of protein folding reactions. As shown for the unfolding of Mb, the multi-probe kinetic measurements will be a powerful tool for investigating the mechanism of folding, in particular for characterizing structural kinetic intermediates. The dynamics of local fluctuations of a well-defined part of RNase S can be monitored by NMR measurements of NH proton exchange. An increasing number of experimental and theoretical studies are focussing on the problem of protein dynamics. Application of NMR methods to protein folding should give extensive information about the structure of intermediates, which cannot be given by other techniques.