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

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