Abstract
The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.
Highlights
Protein folding and unfolding have been extensively studied for several decades, it still attracts numerous researchers’ attention nowadays
Effects of urea and guanidine hydrochloride (GdnHCl) on the activity of protein tyrosine phosphatase (PTPase) To explore the effects of urea and GdnHCl on the enzymatic activity of PTPase, the relative residual activities of PTPase in the presence of different concentrations of urea and GdnHCl were measured, as presented in Fig. 2A&B, respectively
PTPase activity was almost completely lost in 9 M urea or 1 M GdnHCl, indicating that the conformation of the active sites of PTPase almost have been completely changed by these denaturants
Summary
Protein folding and unfolding have been extensively studied for several decades, it still attracts numerous researchers’ attention nowadays. Some non-native states (such as molten globule state) with specific spectroscopic properties distinct from those of native and completely unfolded states have been observed under mildly denaturing conditions [3,4,5]. These conformational states are widely present and result in protein non-cooperative unfolding transitions. Lots of PTPases structures have been resolved to understand its substrate specificity, catalytic mechanism and biologic functions in vivo since the first purification of PTPase in 1988 [11]. Understanding the relationship between PTPase structure, enzymatic activity, folding mechanism and their functions in vivo is critical to better utilize PTPases as therapeutic targets for human diseases. We studied the inactivation kinetics and unfolding processes of PTPase in the presence of urea and GdnHCl to explore the effects of these denaturants on the activity, secondary/tertiary structure and unfolding state of PTPase
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