Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology , TU Dortmund , Otto-Hahn-Strasse 4a , 44227 Dortmund , Germany.
Departments of Biochemistry and Molecular Genetics, Faculty of Medicine , University of Toronto , Toronto , Ontario M5S 1A8 , Canada.
J Am Chem Soc. 2019 May 8;141(18):7347-7354. doi: 10.1021/jacs.8b13636. Epub 2019 Apr 23.
Biomolecular condensates can be functional (e.g., as membrane-less organelles) or dysfunctional (e.g., as precursors to pathological protein aggregates). A major physical underpinning of biomolecular condensates is liquid-liquid phase separation (LLPS) of proteins and nucleic acids. Here we investigate the effects of temperature and pressure on the LLPS of the eye-lens protein γ-crystallin using UV/vis and IR absorption, fluorescence spectroscopy, and light microscopy to characterize the mesoscopic phase states. Quite unexpectedly, the LLPS of γ-crystallin is much more sensitive to pressure than folded states of globular proteins. At low temperatures, the phase-separated droplets of γ-crystallin dissolve into a homogeneous solution at as low as ∼0.1 kbar whereas proteins typically unfold above ∼3 kbar. This observation suggests, in general, that organisms thriving under high-pressure conditions in the deep sea, with pressure of up to 1 kbar, have to cope with this pressure sensitivity of biomolecular condensates to avoid detrimental impacts to their physiology. Interestingly, our experiments demonstrate that trimethylamine- N-oxide, an osmolyte upregulated in deep-sea fish, significantly enhances the stability of the condensed protein droplets, pointing to a previously unrecognized aspect of the adaptive advantage of increased concentrations of osmolytes in deep-sea organisms. As the birth place of life on earth could have been the deep sea, studies of pressure effects on LLPS as presented here are relevant to the possible formation of protocells under prebiotic conditions. A physical framework to conceptualize our observations and further ramifications of biomolecular LLPS at low temperatures and high hydrostatic pressures is discussed.
生物分子凝聚物可以是功能性的（例如，作为无膜细胞器），也可以是功能失调的（例如，作为病理性蛋白质聚集体的前体）。生物分子凝聚物的一个主要物理基础是蛋白质和核酸的液-液相分离（LLPS）。在这里，我们使用紫外/可见和红外吸收、荧光光谱和相差显微镜来研究温度和压力对眼晶状体蛋白 γ-晶体蛋白 LLPS 的影响，以表征介观相态。出人意料的是，γ-晶体蛋白的 LLPS 对压力的敏感性远高于球状蛋白的折叠状态。在低温下，γ-晶体蛋白的相分离液滴在低至约 0.1 kbar 的压力下溶解成均相溶液，而蛋白质通常在高于约 3 kbar 的压力下展开。这一观察结果表明，一般来说，在深海中高压条件下生存的生物体，压力高达 1 kbar，必须应对生物分子凝聚物对这种压力的敏感性，以避免对其生理产生不利影响。有趣的是，我们的实验表明，三甲胺 N-氧化物，一种在深海鱼类中上调的渗透物，显著增强了凝聚蛋白液滴的稳定性，这表明了深海生物中渗透物浓度增加的适应性优势的一个以前未被认识的方面。由于地球生命的诞生地可能是深海，因此，如本文所述，对 LLPS 压力效应的研究与在原始条件下可能形成原细胞有关。讨论了用于概念化我们的观察结果和生物分子 LLPS 在低温和高静水压力下的进一步影响的物理框架。
