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

Kundu, Bishwajit and Guptasarma, Purnananda (2002) Use of a hydrophobic dye to indirectly probe the structural organization and conformational plasticity of molecules in amorphous aggregates of carbonic anhydrase. Biochemical and biophysical research communications, 293 (1). pp. 572-7. ISSN 0006-291X

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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.

Item Type:	Article
Additional Information:	Copyright of this article belongs to Elsevier Science/Academic Press.
Uncontrolled Keywords:	Protein aggregation; Thermal unfolding; Hydrophobic interactions; Aggregate structures
Subjects:	Q Science > QD Chemistry
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	05 Jan 2012 15:13
Last Modified:	05 Jan 2012 15:13
URI:	http://crdd.osdd.net/open/id/eprint/268

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