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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
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