Biswas Saikat, Kundu Jayanta, Mukherjee Sanjib K, Chowdhury Pramit Kumar
Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
ACS Omega. 2018 Apr 30;3(4):4316-4330. doi: 10.1021/acsomega.7b01864. Epub 2018 Apr 19.
In the living cell, biomolecules perform their respective functions in the presence of not only one type of macromolecules but rather in the presence of various macromolecules with different shapes and sizes. In this study, we have investigated the effects of five single macromolecular crowding agents, Dextran 6, Dextran 40, Dextran 70, Ficoll 70, and PEG 8000 and their binary mixtures on the modulation in the domain separation of human serum albumin using a Förster resonance energy transfer-based approach and the translational mobility of a small fluorescent probe fluorescein isothiocyanate (FITC) using fluorescence correlation spectroscopy (FCS). Our observations suggest that mixed crowding induces greater cooperativity in the domain movement as compared to the components of the mixtures. Thermodynamic analyses of the same provide evidence of crossovers from enthalpy-based interactions to effects dominated by hard-sphere potential. When compared with those obtained for individual crowders, both domain movements and FITC diffusion studies show significant deviations from ideality, with an ideal solution being considered to be that arising from the sum of the contributions of those obtained in the presence of individual crowding agents. Considering the fact that domain movements are local (on the order of a few angstroms) in nature while translational movements span much larger lengthscales, our results imply that the observed deviation from simple additivity exists at several possible levels or lengthscales in such mixtures. Moreover, the nature and the type of deviation not only depend on the identities of the components of the crowder mixtures but are also influenced by the particular face of the serum protein (either the domain I-II or the domain II-III face) that the crowders interact with, thus providing further insights into the possible existence of microheterogeneities in such solutions.
在活细胞中，生物分子不仅在一种类型的大分子存在下发挥各自的功能，而是在存在各种形状和大小不同的大分子的情况下发挥功能。在本研究中，我们使用基于Förster共振能量转移的方法研究了五种单一的大分子拥挤剂（葡聚糖6、葡聚糖40、葡聚糖70、聚蔗糖70和聚乙二醇8000）及其二元混合物对人血清白蛋白结构域分离调节的影响，并使用荧光相关光谱法（FCS）研究了小荧光探针异硫氰酸荧光素（FITC）的平移流动性。我们的观察结果表明，与混合物的组分相比，混合拥挤在结构域运动中诱导了更大的协同性。对此进行的热力学分析提供了从基于焓的相互作用到以硬球势为主导的效应的交叉证据。与单个拥挤剂的情况相比，结构域运动和FITC扩散研究均显示出与理想情况有显著偏差，理想溶液被认为是由在单个拥挤剂存在下获得的贡献之和产生的。考虑到结构域运动本质上是局部的（在几埃的量级），而平移运动跨越更大的长度尺度，我们的结果意味着在这种混合物中，在几个可能的水平或长度尺度上存在观察到的与简单加和性的偏差。此外，偏差的性质和类型不仅取决于拥挤剂混合物组分的特性，还受到拥挤剂与之相互作用的血清蛋白特定面（结构域I-II面或结构域II-III面）的影响，从而为这种溶液中可能存在的微不均匀性提供了进一步的见解。
