Warepam Marina, Sharma Gurumayum Suraj, Dar Tanveer Ali, Khan Md Khurshid Alam, Singh Laishram Rajendrakumar
Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India.
Department of Clinical Biochemistry, University of Kashmir, Srinagar, Jammu & Kashmir, India.
PLoS One. 2014 Oct 14;9(10):e109408. doi: 10.1371/journal.pone.0109408. eCollection 2014.
Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (Km and kcat), thermodynamic stability (Tm and ΔHm) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.
渗透溶质是生物体积累的低分子量有机分子，用于辅助蛋白质正确折叠，并在变性应激条件下保护蛋白质的结构完整性。已知渗透溶质诱导的蛋白质折叠是由渗透溶质与变性/未折叠状态的不利相互作用引起的。渗透溶质与天然状态的相互作用对渗透溶质诱导的蛋白质折叠没有显著贡献。因此，我们研究了蛋白质的不同变性状态（由不同变性剂产生）与渗透溶质相互作用以诱导蛋白质折叠的方式是否不同。我们观察到，从热诱导变性状态获得的蛋白质在渗透溶质辅助下重折叠产生的天然分子比从盐酸胍或尿素诱导变性状态起始重折叠产生的天然分子具有更高的酶活性，这表明在渗透溶质重折叠过程中初始变性状态的结构性质决定了折叠后蛋白质分子的催化效率。这些结论是通过系统测量各种变性状态（由于热、尿素和盐酸胍诱导变性）的核糖核酸酶A在各种渗透溶质存在下重折叠得到的折叠天然蛋白质的酶动力学参数（Km和kcat）、热力学稳定性（Tm和ΔHm）以及二级和三级结构而得出的。
