Mechanisms of evolutionary optimized complex folding reactions

The mechanisms and speed limits of evolutionary optimized complex folding reactions were investigated in this study. The two viral model proteins, foldon and SFVP, have evolved under selective pressure for fast and efficient folding and association. The function of foldon, the C-terminal domain of the trimeric phage T4 protein from the Semliki forest virus, has to cleave itself out of a large polyprotein chain co-translationally during protein synthesis. This promotes the biogenesis of the more C-terminal peptide segments in the viral polyprotein.
This work has revealed that even complex folding reactions like quaternary structure formation in foldon and folding of the two-domain SFVP can occur very fast and efficiently via on-path folding intermediates.
The kinetic and thermodynamic studies on the foldon domain provide the first detailed information on a fast-folding trimeric protein. The foldon domain forms a [beta]-propeller-like structure. All folding and association steps seem to be optimized for rapid formation of a stable trimer. At low protein concentration, the folding reaction shows apparent third-order kinetics. From the compact monomeric intermediate formed within the first milliseconds, the native trimer is formed in two consecutive association reactions with bimolecular rate constants up to 6.10 6 M-1s-1. This is close to the fastest association reactions reported for dimeric proteins. At low protein concentrations, an intermediate species is populated to a small amount at equilibrium. This influences the unfolding kinetics only to a small extent, and does not affect the equilibrium stability at all. With increasing protein concentrations folding becomes independent of the protein concentration, indicating that a first-order folding step from a partially folded trimer to the native protein becomes rate-limiting. The half-time of about 3 ms is comparable to fast-folding small single domain proteins. In contrast to small monomeric proteins, however, intermediates become populated very fast, probably providing suitable interfaces to assist trimerisation. Foldon is the first protein where these interfaces are shown to be formed by [beta]-hairpins. At pH 2, the trimer disintegrates into a monomeric A-state with almost identical fluorescence properties as the monomeric folding intermediate. Thus an equilibrium species very similar to the kinetic intermediate is accessible to NMR studies. The A-state consists of a [beta]-hairpin with the same hydrogen bond pattern as the native trimer, but lacks structure in the N- and C-terminus. The hairpin forms within a few microseconds, and its stability is comparable to designed hairpins in alcohol/water mixtures. With state-of-the-art solution NMR techniques, in particular residual dipolar coupling measurements, new insights on structural preferences in the unfolded and partially folded monomer states were gained.
SFVP folds 1000 times faster than any other two-domain protein investigated so far. Studies with wild-type SFVP demonstrated that an intermediate is populated in GdmC1-induced equilibrium transitions at 10°C. At 25°C, only two states are populated in GdmC1 and in urea. A mutant of SFVP containing an additional fluorescence probe in the N-terminal domain (SFVP F160W) shows identical equilibrium behaviour and very similar folding nd unfolding kinetics. Additional unfolding phases besides the main phase also present in the wild-type protein are observed: a slower one and a faster one. The fast folding phase probably originates from the formation of the N-terminal domain. It is difficult to separate the fast and the main folding phase at low urea concentrations, as the two rate constants become to similar. These results provide further evidence for the folding model of SFVP, where an intermediate is slowly formed from the unfolded state (T=170 ms), and then rapidly converts to the native protein (T=37 ms), so that the intermediate is only populated to a small extent.