School of Chemistry and Biochemistry, Thapar University, Patiala, 147004, India.
Council of Scientific and Industrial Research, Institute of Microbial Technology, Sector 39A, Chandigarh, India.
Arch Biochem Biophys. 2018 Sep 15;654:146-162. doi: 10.1016/j.abb.2018.07.018. Epub 2018 Jul 24.
The role of crowding agents on structure and activities of heme proteins has been established. Analysis of kinetic and thermodynamic parameters measured for CO-dissociation reaction of natively-folded carbonmonoxycytochrome c (NCO) and carbonmonoxymyoglobin (MbCO) at different [GdnHCl] or [Urea] in the presence of crowding agents (dextran 40, dextran 70 and ficoll 70) demonstrate that (i) at low denaturant concentrations, crowder presence enhances the denaturant-mediated restricted dynamics of NCO and MbCO, and (ii) at higher denaturant concentrations, large scale unfolding-fluctuations dominate the dynamics and inclusion of crowder counteracts the structural-fluctuations causing the unfolding of proteins. Thermodynamic analysis of thermal and urea-unfolding curves of cytochrome c (Cyt c) and myoglobin (Mb) measured at different [GdnHCl] in presence of crowding agents reveals that crowder presence counterbalances and strengthens the destabilizing action of GdnHCl on stability of Cyt c and Mb, respectively. This study further demonstrates that the size, shape and concentration of crowding agent modulate the effect of crowder on denaturant-mediated dynamics and thermodynamic stability of heme proteins.
拥挤试剂对血红素蛋白结构和活性的作用已经确立。分析动力学和热力学参数，这些参数是在不同 [GdnHCl] 或 [Urea] 浓度下，以及存在拥挤试剂（葡聚糖 40、葡聚糖 70 和菲可）时，对天然折叠的一氧化碳细胞色素 c (NCO) 和一氧化碳肌红蛋白 (MbCO) 的 CO 解离反应进行测量得到的。结果表明：(i) 在低变性剂浓度下，存在拥挤试剂增强了 NCO 和 MbCO 的变性剂介导的受限动力学；(ii) 在更高的变性剂浓度下，大规模的展开-波动主导动力学，包含拥挤试剂会抵消导致蛋白质展开的结构波动。在存在拥挤试剂的情况下，测量不同 [GdnHCl] 浓度下细胞色素 c (Cyt c) 和肌红蛋白 (Mb) 的热和脲变性曲线的热力学分析表明，拥挤试剂的存在分别抵消和增强了 GdnHCl 对 Cyt c 和 Mb 稳定性的去稳定作用。这项研究进一步表明，拥挤试剂的大小、形状和浓度调节了拥挤试剂对血红素蛋白变性剂介导的动力学和热力学稳定性的影响。
