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Slow irreversible unfolding of Pyrococcus furiosus triosephosphate isomerase: separation and quantitation of conformers through a novel electrophoretic approach.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Slow irreversible unfolding of Pyrococcus furiosus triosephosphate isomerase: separation and quantitation of conformers through a novel electrophoretic approach.
 
Creator Mukherjee, Sourav
Sharma, Swati
Kumar, Sanjeev
Guptasarma, Purnananda
 
Subject QD Chemistry
 
Description The thermostability of hyperthermophile proteins is not easily studied because such proteins tend to be extremely recalcitrant to unfolding. Weeks of exposure to structurally destabilizing conditions are generally required to elicit any evidence of conformational change(s). The main reason for this extreme kinetic stability would appear to be the dominance of local unfolding transitions that occur within different parts of the structures of these molecules; put differently, local sub structural unfolding transitions that occur autonomously and reversibly are thought to fail to cooperate to bring about global unfolding in a facile manner, leading to a low overall observed rate of unfolding. For reasons that are not yet fully understood, unfolding is also reported to occur irreversibly in hyperthermophile proteins. Therefore, conventional experimental approaches are often unsuited to the study of their unfolding. Here, we describe a novel electrophoretic approach that facilitates separation, direct visualization, and quantitation of the folded, partially folded, and unfolded forms of the hyperthermophile protein triosephosphate isomerase from Pyrococcus furiosus, produced in the course of its irreversible structural destabilization by the combined action of heat and chemical agents. Our approach exploits (i) the irreversibility of global unfolding effected by heat and denaturants such as urea or guanidine hydrochloride, (ii) the stability of the native form of the protein to unfolding by the anionic detergent sodium dodecyl sulfate, (iii) the differential susceptibilities of various protein conformations to being bound by SDS, and (iv) the differential electrophoretic migration behavior displayed as a consequence of differential SDS binding.
 
Publisher Elsevier Science
 
Date 2005-12-01
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/164/1/guptasarma2005.pdf
Mukherjee, Sourav and Sharma, Swati and Kumar, Sanjeev and Guptasarma, Purnananda (2005) Slow irreversible unfolding of Pyrococcus furiosus triosephosphate isomerase: separation and quantitation of conformers through a novel electrophoretic approach. Analytical biochemistry, 347 (1). pp. 49-59. ISSN 0003-2697
 
Relation http://www.sciencedirect.com/science/article/pii/S0003269705006093
http://crdd.osdd.net/open/164/