Protein unfolding studies of thiol-proteinase inhibitor from goat (<Emphasis Type="Italic">Capra hircus</Emphasis>) muscle in the presence of urea and GdnHCl as denaturants |
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Authors: | Mohammad Aatif Safikur Rahman Bilqees Bano |
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Institution: | (1) Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, Uttar Pradesh, 202002, India;(2) Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India; |
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Abstract: | In recent years, many advances have been made in the understanding of functional and structural characteristics of protein
evolution from denaturant-based studies that subject the protein to a change in the microenvironment. This paper reports the
chemical denaturation of purified goat muscle cystatin (GMC) a thiol-proteinase inhibitor, using urea and guanidine hydrochloride
(GdnHCl). The subtle conformational changes of GMC were monitored by intrinsic fluorescence, extrinsic fluorescence, and CD
spectroscopic techniques. Further, the activity of GMC as a function of increasing concentration of denaturants was also studied.
It was found that increasing the concentration of GdnHCl significantly enhances the inactivation and unfolding of the inhibitor
(GMC). In urea-induced denaturation, the intrinsic and extrinsic fluorescence intensity reveals significant structural changes
in the inhibitor. Further, it was found that at low concentrations of urea, up to 0.5–1.0 M, there was quenching of fluorescence intensity compared with the native form and a red shift of 5 nm was observed up to 5–8 M. The results presented in this paper suggest that GdnHCl-induced denaturation of GMC follows a simple two-state rule in which
native → denatured state transition occurs in a single step. However denaturation with urea proceeds through an intermediate
or non-native state. |
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