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Biophys J, June 2001, p. 2867-2885, Vol. 80, No. 6
Departments of *Chemistry and Chemical Physics,
Linköping University, SE-581 83 Linköping, Sweden
Two different spin labels,
N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide
(IPSL) and (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)-ethyl)amino)naphtalene-1-sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were
attached to the introduced C79 in human carbonic anhydrase (HCA II) to
probe local structural changes upon unfolding and aggregation. HCA II
unfolds in a multi-step manner with an intermediate state populated
between the native and unfolded states. The spin label IPSL and the
fluorescent label IAEDANS reported on a substantial change in mobility
and polarity at both unfolding transitions at a distance of 7.4-11.2
Å from the backbone of position 79. The shorter and less flexible
labels BADAN and MTSSL revealed less pronounced spectroscopic changes
in the native-to-intermediate transition, 6.6-9.0 Å from the
backbone. At intermediate guanidine (Gu)-HCl concentrations the
occurrence of soluble but irreversibly aggregated oligomeric protein
was identified from refolding experiments. At ~1 M Gu-HCl the
aggregation was found to be essentially complete. The size and
structure of the aggregates could be varied by changing the protein
concentration. EPR measurements and line-shape simulations together
with fluorescence lifetime and anisotropy measurements provided a
picture of the self-assembled protein as a disordered protein structure
with a representation of both compact as well as dynamic and polar
environments at the site of the molecular labels. This suggests that a
partially folded intermediate of HCA II self-assembles by both local
unfolding and intermolecular docking of the intermediates vicinal to
position 79. The aggregates were determined to be 40-90 Å in diameter
depending on the experimental conditions and spectroscopic technique used.
Biophys J, June 2001, p. 2867-2885, Vol. 80, No. 6
© 2001 by the Biophysical Society 0006-3495/01/06/2867/19 $2.00
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