A comparison of experimental and computational methods for mapping the interactions present in the transition state for folding of FKBP12.

The folding pathway of FKBP12, a 107 residue α/β protein, has been characterised in detail using a combination of experimental and computational techniques. FKBP12 follows a two-state model of folding in which only the denatured and native states are significantly populated; no intermediate states are detected. The refolding rate constant in water is 4 s(-1) at 25 °C. Two different experimental strategies were employed for studying the transition state for folding. In the first case, a non-mutagenic approach was used and the unfolding and refolding of the wild-type protein measured as a function of experimental conditions such as temperature, denaturant, ligand and trifluoroethanol (TFE) concentration. These data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The ligand-binding site, whichis mainly formed by two long loops, is largely unstructured in the transition state. TFE experiments suggest that the α-helix may be formed in the transition state. The second experimental approach involved using protein engineering techniques with φ-value analysis. Residue-specific information on the structure and energetics of the transition state can be obtained by this method. 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. In contrast 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 inhibitor 2 and the SH3 domainfrom α-spectrin. For FKBP12, the central three strands of the β-sheet (2, 4 and 5), comprise the most structured region of the transition state. In particular Val 101, which is one of the most highly buried residues and located in the middle of the central β-strand,makes approximately 60% of its native interactions. The outer β-strands, and the ends of the central β-strands are formed to a lesser degree. The short α-helix is largely unstructured in the transition state as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side chains within β-strands 2, 4 and 5 and the C-terminus of the α-helix. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. High-temperature molecular dynamic simulations on the unfoldingpathway of FKBP12 are in good agreement with the experimental results.