Folding pathway of FKBP12 and characterisation of the transition state

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immuno philin), has been characterised. Unfolding and refolding rate constants hav e been determined over a wide range of denaturant concentrations and data a re shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of dena turant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular di chroism experiments in which no fast "burst" phases are observed. FKBP12 wi th 107 residues, is the largest protein to date which folds with simple two -state kinetics in water (k(F) = 4 s(-1) at 25 degrees C). The topological crossing of two loops in FKBP12 a structural element suggested to cause kin etic traps during folding, seems to have little effect on the folding pathw ay. The transition state for folding has been characterised by a series of expe riments on wild-type FKBP12. Information on the thermodynamic nature of, th e solvent accessibility of, and secondary structure in, the transition stat e was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and triflu oroethanol concentration. Ln addition, unfolding and refolding studies in t he presence of Ligand provided information on the structure of the Ligand-b inding pocket in the transition state. The data suggest a compact transitio n state relative to the unfolded state with some 70% of the surface area bu ried. The Ligand-binding site, which is formed mainly by two loops, is larg ely unstructured in the transition state. The trifluoroethanol experiments suggest that the a-helix may be formed in the transition state. These resul ts are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper). (C) 1999 Academic Press .