Department of Physiology & Biophysics, Stony Brook University, Stony Brook, NY 11794-8661, USA.
Department of Physics and Astronomy, Clemson University, Clemson, SC 29634-0978, USA.
J Mol Biol. 2021 Mar 5;433(5):166793. doi: 10.1016/j.jmb.2020.166793. Epub 2021 Jan 1.
Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.
许多蛋白质由独立折叠的结构域通过柔性连接体连接而成。这些连接体的一级序列和长度可以设定连接结构域的有效浓度，从而影响缔合速率和酶活性。这些连接体的长度可能对环境条件敏感，这就提出了一个问题，即在稀缓冲液中的研究如何与高度拥挤的细胞环境相关。为了研究连接体在结构域分离中的作用，我们测量了一系列串联荧光蛋白的荧光蛋白-荧光共振能量转移（FP-FRET），这些串联荧光蛋白的结构域之间的连接体长度不同。我们使用离散分子动力学来绘制潜在的构象分布，这揭示了我们通过单分子 FRET 来证实的分子内接触状态。模拟发现，连接的荧光蛋白增加了连接体的长度，并减缓了构象动力学，与裸露的连接体相比。这使得 CLYs 成为固有连接体特性的不良传感器。然而，我们还表明，CLYs 中的 FP-FRET 对溶剂质量和大分子拥挤度敏感，使它们成为有效的环境传感器。最后，我们将相同的蛋白质靶向到活哺乳动物细胞的质膜，以测量细胞内的 FP-FRET。当与质膜连接时，测量的 FP-FRET 与在稀缓冲液中相同。虽然在光物理方面仍然存在一些注意事项，但这表明这些 CLY 蛋白的超三级构象整体可能不受这种特定细胞环境的影响。
