Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
Center of Excellence in Biotechnology Research, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; Protein Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
Int J Biol Macromol. 2021 Jun 30;181:552-560. doi: 10.1016/j.ijbiomac.2021.03.089. Epub 2021 Mar 17.
Macromolecular crowding can have significant consequences on the structure and dynamics of a protein. The size and shape of a co-solute molecule and the nature of protein contribute significantly in macromolecular crowding, which results in different outcomes in similar conditions. The structure of apo-myoglobin (apo-Mb) both in the absence and presence of denaturants (GdmCl and urea) was investigated in crowded conditions at pH 7.0, with a comparable size of crowders (~70 kDa) but of different shapes (ficoll and dextran) at various concentrations using spectroscopic techniques like absorption and circular dichroism to monitor changes in secondary and tertiary structure, respectively. The crowders in the absence of denaturants showed structural stabilization of the tertiary structure while no significant change in the secondary structure was observed. The effect of crowders on the stability of the protein was also investigated using probes such as Δε and θ using chemical denaturants. The analysis of chemical-induced denaturation curves showed that both the crowders stabilize apo-Mb by increasing the values of the midpoint of transition (C) and change in free energy in the absence of denaturant (∆G°), and it was observed that dextran 70 shows more stabilization than ficoll 70 under similar conditions. In this study apo-Mb showed stabilization under crowded conditions, which is a deviation from earlier work from our group where holo form of the same protein was destabilized. This study emphasizes that volume exclusion is a dominant force in a simple protein while soft interactions may play important role in the proteins that are possessing prosthetic group. Hence, the effect of crowders is protein-dependent, and excluded volume plays a great role in the stabilization of apo-Mb, which does not interact with the crowders.
大分子拥挤会对蛋白质的结构和动力学产生重大影响。共溶剂分子的大小和形状以及蛋白质的性质在大分子拥挤中起着重要作用，这导致在相似条件下产生不同的结果。在 pH 7.0 时，在拥挤条件下，使用吸收和圆二色性等光谱技术研究了apo-肌红蛋白（apo-Mb）在缺乏和存在变性剂（GdmCl 和尿素）时的结构，共溶剂的大小相当（~70 kDa），但形状不同（ficoll 和 dextran），浓度不同。在没有变性剂的情况下，共溶剂显示出三级结构的结构稳定性，而二级结构没有观察到明显变化。还使用探针如Δε和θ研究了共溶剂对蛋白质稳定性的影响使用化学变性剂。化学诱导变性曲线的分析表明，共溶剂通过增加转变中点（C）的值和在没有变性剂的情况下自由能的变化（∆G°）来稳定 apo-Mb，并且观察到在相似条件下 dextran 70 比 ficoll 70 显示出更多的稳定性。在这项研究中，apo-Mb 在拥挤条件下显示出稳定性，这与我们小组早期的工作不同，在早期工作中，相同蛋白质的全酶形式被不稳定化。这项研究强调，体积排阻是简单蛋白质中的主要力，而软相互作用可能在具有辅基的蛋白质中发挥重要作用。因此，共溶剂的影响是蛋白质依赖性的，排除体积在 apo-Mb 的稳定中起着重要作用，apo-Mb 不与共溶剂相互作用。
