Formation of local asymmetric structures in the slow folding reaction
of human carbonic anhydrase II

Dick Andersson, IFM Chemistry Linkopings University S-581 83 SWEDEN

In the study of protein folding we use human carbonic anhydrase II
(HCA II) as a model protein. Circular dichroism (CD) and fluorescence
have been used to monitor the folding process in engineered tryptophan
mutants of HCA II. CD kinetic measurements on engineered tryptophan
mutants of HCA II in the near- UV region have been used to probe the
development of asymmetric environments around specific Trp residues
during the refolding of HCA II. The formation of the active site was
probed by the binding of a fluorescent sulfonamide inhibitor. So far
three of the seven Trp residues in HCA II have been analyzed. The
three selected Trp residues are situated in different sites of the
protein and they are the major contributors to the near-UV CD
spectrum. The aim of the study is to describe the formation of
specific native structures around individual sites during the
refolding process of HCA II. The refolding kinetics for the studied
Trp residues shows that the asymmetric environment around these Trp
residues is formed at different rates, showing that the formation of
native-like tertiary structure occurs with varying rates in different
regions of the protein.

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