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of human carbonic anhydrase II has been charact erized by use of seven variants in which tryptophan residues have been replaced by Phe or Cys, in each case giving proteins with six tryptop hans. Intrinsic tryptophan fluorescence was used to monitor the refold ing in the 2 ms-60 s time range, and kinetic traces showing the contri butions from each particular tryptophan were obtained by calculation o f differences between the wild-type protein and the variants. Earlier assignment [Martensson, L.-G., Jonasson, P., Freskgard, P.-O., Svensso n, M., Carlsson, U., & Jonsson, B.-H. (1995) Biochemistry, 34, 1011-10 21] of specific fluorescence properties to each tryptophan, especially regarding energy transfer and intrinsic fluorescence quenching, has m ade it possible to use the kinetic data to describe the formation of t ertiary structure at defined tryptophan residues. In summary, it was f ound that tertiary structure is formed earlier at those tryptophans th at are associated with the central core of beta-strands than at trypto phan residues in the N-terminal minidomain.