Tertiary structure formation at specific tryptophan side chains in the refolding of human carbonic anhydrase II.

The refolding reaction of human carbonic anhydrase II has been characterized by use of seven variants in which tryptophan residues have been replaced by Phe or Cys, in each case giving proteins with six tryptophans. Intrinsic tryptophan fluorescence was used to monitor the refolding in the 2 ms-60 s time range, and kinetic traces showing the contributions from each particular tryptophan were obtained by calculation of differences between the wild-type protein and the variants. Earlier assignment [Mårtensson, L.-G., Jonasson, P., Freskgard, P.-O., Svensson, M., Carlsson, U., & Jonsson, B.-H. (1995) Biochemistry 34, 1011-1021] of specific fluorescence properties to each tryptophan, especially regarding energy transfer and intrinsic fluorescence quenching, has made it possible to use the kinetic data to describe the formation of tertiary structure at defined tryptophan residues. In summary, it was found that tertiary structure is formed earlier at those tryptophans that are associated with the central core of beta-strands than at tryptophan residues in the N-terminal minidomain.