Mapping the interactions present in the transition state for unfolding/folding of FKBP12

The structure of the transition state for folding/unfolding of the immunoph ilin FKBP12 has been characterised using a combination of protein engineeri ng techniques, unfolding kinetics, and molecular dynamics simulations. A to tal of 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. The tra nsition state for folding is compact compared with the unfolded state, with an approximately 30 % increase in the native solvent-accessible surface ar ea. All of the interactions are substantially weaker in the transition stat e, as probed by both experiment and molecular dynamics simulations. In cont rast to some other proteins of this size, no element of structure is fully formed in the transition state; instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhib itor 2 and the SH3 domain from alpha-spectrin. For FKBP12 the central three strands of the beta-sheet, beta-strand 2, beta-strand 4 and beta-strand 5, comprise the most structured region of the transition state. In particular Val101, which is one of the most highly buried residues and located in the middle of the central beta-strand, makes approximately 60 % of its native interactions. The outer beta-strands and the ends of the central beta-stran ds are formed to a lesser degree. The short alpha-helix is largely unstruct ured in the transition state, as are the loops. The data are consistent wit h a nucleation-condensation model of folding, the nucleus of which is forme d by side-chains within beta-strands 2, 4 and 5, and the C terminus of the alpha-helix. The precise residues involved in the nucleus differ in the two simulated transition state ensembles, but the interacting regions of the p rotein are conserved. These residues are distant in the primary sequence, d emonstrating the importance of tertiary interactions in the transition stat e. The two independently derived transition state ensembles are structurall y similar, which is consistent with a Bronsted analysis confirming that the transition state is an ensemble of states close in structure. (C) 1999 Aca demic Press.