Folding pathway of FKBP12 and characterisation of the transition state

  • Main E
  • Fulton K
  • Jackson S
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Abstract

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are 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 denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast 'burst' phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (k(F) = 4 s-1at 25°C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway. The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the α-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).

Author-supplied keywords

  • FKBP12
  • Immunophilin
  • Protein folding
  • Transition state
  • Two-state folding

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