Stoneman Michael R, Yokoi Koki, Biener Gabriel, Killeen Thomas D, Adhikari Dhruba P, Rahman Sadia, Harikumar Kaleeckal G, Miller Laurence J, Raicu Valerică
Department of Physics, University of Wisconsin-Milwaukee, WI 53211, USA.
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ, USA.
Res Sq. 2024 Jul 19:rs.3.rs-4683780. doi: 10.21203/rs.3.rs-4683780/v1.
The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.
蛋白质在活细胞中相互作用形成短暂四级结构的功能意义再怎么强调也不为过。然而，目前的方法无法获取四级结构信息，也无法在活细胞内确定结构。G蛋白偶联受体（GPCR）的动态多样性、丰度和功能多样性给现有技术带来了无数挑战，但也使这些蛋白质成为研究活细胞中受体-配体、受体-受体和受体-下游效应器界面复杂相互调节的新技术的理想试验台。在此，我们展示了一种新方法的开发和应用，该方法通过将活细胞中荧光标记蛋白聚集成寡聚体复合物群体的FRET效率分布（或光谱图）与分子动力学（MD）模拟获得的FRET供体和受体的扩散样轨迹相结合，能够克服现有挑战。我们的方法提供了活细胞中人促胰液素受体（hSecR）寡聚体内结合界面的原子水平图像，并允许提取关于GPCR寡聚化功能的机制性见解。这种FRET-MD光谱方法是研究蛋白质-蛋白质结合机制的强大平台，为研究活细胞中任何膜蛋白的稳定以及短暂四级结构开辟了新途径。
