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EXCIMER AND MONOMER FLUORESCENCE OF PYRENE LABELED HUMAN CARBONIC ANHYDRASE II Detection of residual structure during strong denaturing conditions P. Hammarström.1), B. Kalman1), U. Carlsson1), and B.-H Jonsson2) 1)IFM/Dept. of Chemistry, Linköping University, Linköping, Sweden, E-mail: ham@ifm.liu.se 2)Dept. of Biochemistry , Umeå University, Umeå, Sweden To be able to deduce the folding pathway of proteins much effort has been set on investigating intermediate structures, found at various stages during folding. Previous studies in our laboratory on human carbonic anhydrase II, HCAII, has indicated a very stable core in the protein (1). Such a stable core can presumably act as a seed in the folding process and could therefore be assumed to be an early intermediate. The stability of the central part of HCAII was therefore the target for further investigation. In the present work a novel approach for studying folding and stability of HCAII was employed. Fluorescent pyrene probes were attached to cysteine residues introduced by site directed mutagenesis to various positions in the structure. If two pyrenyl moieties are within a few Å distance and have the correct relative direction they can form an excimer upon excitation. In the native state of HCAII attachment of pyrene probes at positions in *-strand 3 and 7 which are situated in the central part of HCAII display an excimer band in the fluorescence spectrum. Unfolding of the protein, however, separates the two sites and a pure pyrene monomer spectra is displayed. The disappearance of the excimer band reveal an unfolding transition at conditions far more denaturing than what is considered to be the global unfolding of the protein as revealed by CD and tryptophan fluorescence. We therefore conclude that residual structure exist and that the central part of HCAII in the "unfolded state" is compact. (1) Svensson, M. et al., 1995, Biochemistry 34, 8606 ## Back to the list of abstracts