Reversible unfolding of Escherichia coli alkaline phosphatase: active site can be reconstituted by a number of pathways.
Acid-induced and guanidine hydrochloride (GdnCl)-induced reversible unfolding of Escherichia coli alkaline phosphatase (AP) was characterized under equilibrium conditions. The protein was exposed to extreme conditions of pH 2.0 or 6 M GdnCl and was subsequently returned to normal conditions. Associated changes in the protein structure was probed by various spectroscopic methods. The changes in the functional properties were monitored by measuring enzymatic activity, capacity to renature spontaneously upon removal of the denaturant, and renaturation in presence of various site-specific and nonspecific effector molecules, in the absence and presence of beta-mercaptoethanol. Analysis of the fluorescence and CD spectra showed that the unfolding of the organized structures was much more extensive in 6 M GdnCl than at pH 2.0. Intrachain S-S bonds in each unfolded state were accessible to reduction by beta-mercaptoethanol. The effectors Zn2+ and ATP induced renaturation of active site only under reducing conditions, whereas Triton X-100 or alpha-crystallin needed the presence of some organized structure. The reconstituted protein from each denatured state without or with an effector showed different CD spectra. It is concluded that the active site domain of AP could be reconstituted independently of other structural domains in different pathways.
Sarkar
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Department of Biochemistry and Biophysics, University of Kalyani, India.
Ghosh
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D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006146:Guanidines; C027132:1-anilino-8-naphthalenesulfonate; D000469:Alkaline Phosphatase; D019791:Guanidine