title: Slow irreversible unfolding of Pyrococcus furiosus triosephosphate isomerase: separation and quantitation of conformers through a novel electrophoretic approach.
abstract: 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.
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Sourav
authors.initials: S
authors.lastname: Mukherjee
authors.forename: Swati
authors.initials: S
authors.lastname: Sharma
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Kumar
authors.forename: Purnananda
authors.initials: P
authors.lastname: Guptasarma
mesh_terms: D002942:Circular Dichroism; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D020027:Pyrococcus furiosus / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000302:isolation & purification*; D014508:Urea
chemical_list: D012967:Sodium Dodecyl Sulfate; D014508:Urea; D014305:Triose-Phosphate Isomerase; D019791:Guanidine