Department of Biochemistry, Aligarh Muslim University, Uttar Pradesh, India.
Department of Biochemistry, Aligarh Muslim University, Uttar Pradesh, India.
Int J Biol Macromol. 2018 Jan;106:20-29. doi: 10.1016/j.ijbiomac.2017.07.172. Epub 2017 Aug 5.
Cysteine proteinase inhibitors play an essential role in maintaining the proper functioning of all living cells by virtue of its thiol protease regulatory properties. Chemical denaturation of a new variant of cystatin super family has been studied by various biophysical techniques in order to characterize the unfolded and denatured state. Denaturation of garlic phytocystatin (GPC) has been investigated using urea and guanidine hydrochloride (GdnHCl). Different biophysical techniques such as intrinsic fluorescence, circular dichroism and FTIR exhibited an altered structure of garlic phytocystatin with increasing concentration of denaturant. The inhibitory activity of GPC decreases with increasing concentration of denaturant. Increased fluorescence intensity along with red shift reflects the unfolding of GPC at higher concentration of denaturant. GdnHCl induced unfolding showed presence of indiscernible intermediate as followed by ANS binding studies. However, denaturation by urea did not show any intermediates. Mid-point transition was observed at 4.7±0.1M urea and 2.32±0.1M GdnHCl. Circular dichroism and FTIR results indicate the 50% loss of secondary structure at 5M urea and 2.5M GdnHCl. This study provides intriguing insight into the possible alteration of structure, stability and function of GPC induced by urea and GdnHCl.
半胱氨酸蛋白酶抑制剂通过其硫醇蛋白酶调节特性，在维持所有活细胞的正常功能方面发挥着重要作用。为了表征展开和变性状态，已经使用各种生物物理技术研究了半胱氨酸蛋白酶抑制剂超家族的新变体的化学变性。已经使用脲和盐酸胍（GdnHCl）研究了大蒜植物半胱氨酸蛋白酶抑制剂（GPC）的变性。不同的生物物理技术，如内源荧光、圆二色性和 FTIR，随着变性剂浓度的增加，显示出大蒜植物半胱氨酸蛋白酶抑制剂结构的改变。GPC 的抑制活性随变性剂浓度的增加而降低。荧光强度的增加伴随着红移反映了在较高浓度的变性剂下 GPC 的展开。GdnHCl 诱导的展开表明存在不可识别的中间产物，如随后的 ANS 结合研究所示。然而，脲变性没有显示任何中间产物。在 4.7±0.1M 脲和 2.32±0.1M GdnHCl 处观察到中点转变。圆二色性和 FTIR 结果表明，在 5M 脲和 2.5M GdnHCl 下，二级结构的 50%损失。这项研究提供了有趣的见解，即脲和 GdnHCl 可能会改变 GPC 的结构、稳定性和功能。
