Transition state heterogeneity in GCN4 coiled coil folding studied by using multisite mutations and crosslinking

We have investigated the folding behavior of dimeric and covalently crossli nked versions of the 33-residue cc-helical GCN4-pl coiled coil derived from the leucine zipper region of the transcriptional activator GCN4, The effec ts of multisite substitutions indicate that folding occurs along multiple r outes with nucleation sites located throughout the protein. The similarity in activation energies of the different routes together with an analysis of intrinsic helical propensities indicate that minimal helix is present befo re a productive collision of the two chains. However, approximately one-thi rd to one-half of the total helical structure is formed in the postcollisio n transition state ensemble. For the crosslinked, monomeric version, foldin g occurs along a single robust pathway. Here, the region nearest the crossl ink, with the least helical propensity, is structured in the transition sta te whereas the region farthest from the tether, with the most propensity, i s completely unstructured. Hence, the existence of transition state heterog eneity and the selection of folding routes critically depend on chain topol ogy.