Polyethylene glycol (PEG) is a flexible, nontoxic polymer commonly used in biological and medical research, and it is generally regarded as biologically inert. PEG molecules of variable sizes are also used as crowding agents to mimic intracellular environments. A recent study with PEG crowders revealed decreased catalytic activity of Escherichia coli prolyl-tRNA synthetase (Ec ProRS), where the smaller molecular weight PEGs had the maximum impact. The molecular mechanism of the crowding effects of PEGs is not clearly understood. PEG may impact protein conformation and dynamics, thus its function. In the present study, the effects of PEG molecules of various molecular weights and concentrations on the conformation and dynamics of Ec ProRS were investigated using a combined experimental and computational approach including intrinsic tryptophan fluorescence spectroscopy, atomic force microscopy, and atomistic molecular dynamic simulations. Results of the present study suggest that lower molecular weight PEGs in the dilute regime have modest effects on the conformational dynamics of Ec ProRS but impact the catalytic function primarily via the excluded volume effect; they form large clusters blocking the active site pocket. In contrast, the larger molecular weight PEGs in dilute to semidilute regimes have a significant impact on the protein’s conformational dynamics; they wrap on the protein surface through noncovalent interactions. Thus, lower-molecular-weight PEG molecules impact protein dynamics and function via crowding effects, whereas larger PEGs induce confinement effects. These results have implications for the development of inhibitors for protein targets in a crowded cellular environment.

中文翻译：

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聚乙二醇通过拥挤和限制效应影响大肠杆菌脯氨酰-tRNA 合成酶的构象和动力学

聚乙二醇（PEG）是一种柔性、无毒的聚合物，常用于生物和医学研究，通常被认为具有生物惰性。不同大小的 PEG 分子也被用作拥挤剂来模拟细胞内环境。最近的一项 PEG 拥挤器研究表明，大肠杆菌脯氨酰-tRNA 合成酶 (Ec ProRS) 的催化活性降低，其中较小分子量的 PEG 影响最大。 PEG 拥挤效应的分子机制尚不清楚。 PEG 可能会影响蛋白质的构象和动力学，从而影响其功能。在本研究中，使用本征色氨酸荧光光谱、原子力显微镜和原子分子动力学模拟等实验和计算相结合的方法，研究了不同分子量和浓度的PEG分子对Ec ProRS的构象和动力学的影响。本研究的结果表明，稀释状态下的较低分子量 PEG 对 Ec ProRS 的构象动力学影响不大，但主要通过排除体积效应影响催化功能；它们形成大簇，阻塞活性位点袋。相比之下，稀释至半稀释状态下的较大分子量 PEG 对蛋白质的构象动力学具有显着影响。它们通过非共价相互作用包裹在蛋白质表面。因此，较低分子量的 PEG 分子通过拥挤效应影响蛋白质动力学和功能，而较大的 PEG 则会产生限制效应。这些结果对于拥挤细胞环境中蛋白质靶点抑制剂的开发具有重要意义。