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Use of a hydrophobic dye to indirectly probe the structural organization and conformational plasticity of molecules in amorphous aggregates of carbonic anhydrase.

Abstract：

:Understanding protein aggregation may hold important clues to understanding what goes wrong with protein folding in neurodegenerative disorders and in bioreactors in which proteins are overexpressed. Unfortunately, aggregates tend to be intractable to most standard methods of biochemical investigation. Thus, relatively little is even now known about the micro- and macro-structural features of aggregates. To gain insights into the thermal aggregation of a model globular protein [bovine carbonic anhydrase (BCA)], we have used spectrofluorimetry to examine the binding of a hydrophobic dye, 8-anilinonaphthalene sulfonate (ANS), to hydrophobic clusters on the protein's surface both before and after heat-induced aggregation and upon cooling. Whereas native BCA shows no surface hydrophobicity, thermally aggregated BCA displays significant hydrophobicity both in the heated state and upon cooling. The timing of the addition of ANS in the course of aggregation makes no net difference to the ANS bound; we argue that this suggests that aggregates are essentially porous. Cooling of aggregates results in a dramatic, fully reversible increase in ANS binding that cannot be explained by the temperature dependence of fluorescence quantum yield alone; we argue that the enhancement of fluorescence upon cooling indicates possible structural consolidation of unfolded regions within aggregates (akin to refolding), with the required structural reorganization being facilitated by porosity. Finally, implications of porosity in aggregates are discussed, in particular, for the possible immobilization of enzymes through fusion with aggregation-prone protein domains.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Kundu B,Guptasarma P

doi

10.1016/S0006-291X(02)00257-7

subject

Has Abstract

pub_date

2002-04-26 00:00:00

pages

572-7

issue

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(02)00257-7

journal_volume

293

pub_type

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Use of a hydrophobic dye to indirectly probe the structural organization and conformational plasticity of molecules in amorphous aggregates of carbonic anhydrase.