Abstract
The role of domains in defining the equilibrium and kinetic folding properties of dihydrofolate reductase (DHFR) from Escherichia coli was probed by examining the thermodynamic and kinetic properties of a set of variants in which the chain connectivity in the discontinuous loop domain (DLD) and the adenosine-binding domain (ABD) was altered by permutation. To test the concept that chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident, permutations were introduced at one position within the ABD, one within the DLD and one at a boundary between the domains. The results demonstrated that a continuous ABD is required for a stable thermal intermediate and a continuous DLD is required for a stable urea intermediate. The permutation at the domain interface had both a thermal and urea intermediate. Strikingly, the observable kinetic folding responses of all three permuted proteins were very similar to the wild-type protein. These results demonstrate a crucial role for stable domains in defining the energy surface for the equilibrium folding reaction of DHFR. If domain connectivity affects the kinetic mechanism, the effects must occur in the sub-millisecond time range.