Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center, Russian Academy of Sciences, Lobachevsky Str. 2/31, Kazan 420111, Russia.
Institute of Chemistry, Kazan Federal University, Kremlevskaya Str. 18, Kazan 420008, Russia.
Int J Mol Sci. 2023 Jul 6;24(13):11148. doi: 10.3390/ijms241311148.
Intracellular environment includes proteins, sugars, and nucleic acids interacting in restricted media. In the cytoplasm, the excluded volume effect takes up to 40% of the volume available for occupation by macromolecules. In this work, we tested several approaches modeling crowded solutions for protein diffusion. We experimentally showed how the protein diffusion deviates from conventional Brownian motion in artificial conditions modeling the alteration of medium viscosity and rigid spatial obstacles. The studied tracer proteins were globular bovine serum albumin and intrinsically disordered α-casein. Using the pulsed field gradient NMR, we investigated the translational diffusion of protein probes of different structures in homogeneous (glycerol) and heterogeneous (PEG 300/PEG 6000/PEG 40,000) solutions as a function of crowder concentration. Our results showed fundamentally different effects of homogeneous and heterogeneous crowded environments on protein self-diffusion. In addition, the applied "tracer on lattice" model showed that smaller crowding obstacles (PEG 300 and PEG 6000) create a dense net of restrictions noticeably hindering diffusing protein probes, whereas the large-sized PEG 40,000 creates a "less restricted" environment for the diffusive motion of protein molecules.
细胞内环境包括在受限介质中相互作用的蛋白质、糖和核酸。在细胞质中，排除体积效应占据了大分子可占据体积的 40%。在这项工作中，我们测试了几种模拟拥挤溶液中蛋白质扩散的方法。我们通过模拟介质粘度变化和刚性空间障碍物的人工条件，实验证明了蛋白质扩散如何偏离传统的布朗运动。研究的示踪蛋白是球状牛血清白蛋白和天然无序的α-酪蛋白。使用脉冲场梯度 NMR，我们研究了不同结构的蛋白质探针在均相（甘油）和非均相（PEG 300/PEG 6000/PEG 40,000）溶液中的平移扩散，作为 crowder 浓度的函数。我们的结果表明，均匀和非均匀拥挤环境对蛋白质自扩散有根本不同的影响。此外，所应用的“格点上的示踪剂”模型表明，较小的拥挤障碍物（PEG 300 和 PEG 6000）形成了一个明显阻碍扩散蛋白质探针的密集限制网络，而较大尺寸的 PEG 40,000 为蛋白质分子的扩散运动创造了一个“限制较少”的环境。
