Biotechnology Division, Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India.
Eur Biophys J. 2010 Sep;39(10):1385-96. doi: 10.1007/s00249-010-0593-z. Epub 2010 Mar 24.
Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has alpha-helix and beta-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.
动力学稳定的同源二聚体丝氨酸蛋白酶 milin 表现出对温度、pH 值和变性剂（尿素、盐酸胍 (GuHCl) 和异硫氰酸胍 (GuSCN)）的高构象稳定性，通过圆二色性、荧光、差示扫描量热法和活性测量进行探测。GuSCN 诱导 milin 完全展开，而温度、尿素和 GuHCl 即使在低 pH 值下也仅诱导部分展开，通过具有不同特征的几个中间态。其中一些中间态具有部分活性（即在 pH 7.0 时的尿素和 2 M GuHCl 中），并且一些中间态也表现出强烈的 ANS 结合。从与平衡展开实验耦合的本征荧光测量可以明显看出，蛋白质中的所有三个色氨酸都埋藏在刚性、紧凑的核心中。蛋白质作为二聚体展开，其中展开事件先于二聚体解离，这通过流体力学研究得到证实。这里进行的溶液研究以及以前的生化特性表明，该蛋白质具有富含 alpha-螺旋和 beta-折叠的区域或结构域，这些区域或结构域独立展开，单体的缔合是同源的。已经对 milin 的复杂展开途径和中间态进行了表征。讨论了结果在物理、生理和可能的治疗方面的重要性。
