The folding of a 98 residue protein, muscle acylphosphatase (AcP), has been studied using a variety of techniques including circular dichroism, fluorescence and NMR spectroscopy following transfer of chemically denatured protein into refolding conditions. A low-amplitude phase, detected in concurrence with the main kinetic phase, corresponds to the folding of a minor population (13%) of molecules with one or both proline residues in a cis conformation, as shown from the sensitivity of its rate to peptidyl prolyl isomerase. The major phase of folding has the same kinetic characteristics regardless of the technique employed to monitor it. The plots of the natural logarithms of folding and unfolding rate constants versus urea concentration are linear over a broad range of urea concentrations. Moreover, the initial state formed rapidly after the initiation of refolding is highly unstructured, having a similar circular dichroism, intrinsic fluorescence and NMR spectrum as the protein denatured at high concentrations of urea. All these results indicate that AcP folds in a two-state manner without the accumulation of intermediates. Despite this, the folding of the protein is extremely slow. The rate constant of the major phase of folding in water, kfH2O, is 0.23 s-1 at 28 degreesC and, at urea concentrations above 1 M, the folding process is slower than the cis-trans proline isomerisation step. The slow refolding of this protein is therefore not the consequence of populated intermediates that can act as kinetic traps, but arises from a large intrinsic barrier in the folding reaction.
Copyright 1998 Academic Press.