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

Rop is a four-helix-bundle protein formed by the association of two helix-loop-helix monomers. The short helix-connecting loop was replaced with a series of polyglycine linkers of increasing length. These mutant proteins all appear to fold via the same general mechanism as that of the wild-type protein, even at the longest loop lengths. Replacement of the wild-type two-residue loop (Asp-Ala) with a (Gly-Gly) linker accelerates both unfolding and refolding rates. These changes in folding and unfolding kinetics likely indicate 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 intermediate. These dependences underscore the importance of loop closure and help refine the model for Rop's folding, implicating a dimeric intermediate involving hairpin formation. These observations show that loop alteration may be useful as a general technique for dissecting protein folding pathways.