Using loop length variants to dissect the folding pathway of a four-helix-bundle protein

Rop is a few-helix-bundle protein formed by the association of two helix-lo op-helix monomers. The short helix-connecting loop was replaced with a seri es of polyglycine Linkers of increasing length. These mutant proteins all a ppear to fold via the same general mechanism as that of the wild-type prote in, even at the longest loop lengths. Replacement of the wild-type two-resi due loop (Asp-Ala) with a (Gly-Gly) linker accelerates both unfolding and r efolding rates. These changes in folding and unfolding kinetics likely indi cate an alteration in the energy of the transition state. As the length of the glycine linker is further increased, the unfolding rate increases while the refolding rates decrease. The influence of loop length is not limited to these rates, but also impacts upon the stability of the folding intermed iate. These dependences underscore the importance of loop closure and help refine the model for Rep's folding, implicating a dimeric intermediate invo lving hairpin formation. These observations show that loop alteration may b e useful as a general technique for dissecting protein folding pathways. (C ) 1999 Academic Press.