Probing the Dimerization of Transmembrane Helices in Eukaryotic and Lipid Bilayer Membranes

细胞膜中疏水跨膜（TM）螺旋之间的横向相互作用是多跨膜蛋白折叠和受体酪氨酸激酶（RTKs）介导的信号转导的基础。由于当前实验方法的许多限制，解码这些过程所需的膜中二聚化能量的定量测量具有挑战性。PI将开发新的通用方法的研究TM螺旋二聚化在生物膜。PI将在真核生物膜中进行测量，并引入FRET-Western测定，在天然细胞膜的背景下探测膜蛋白的稳定性和结构。PI还将开发表面支持的脂质双分子层，通过整合脂质组成的不对称性和蛋白质的单向取向来模拟质膜。这项工作将揭示复杂的质膜环境在膜蛋白相互作用中的作用。FRET-Western将允许识别改变TM二聚体稳定性或TM二聚体结构的TM结构域突变，从而为膜中蛋白质二聚化的结构决定因素提供新的见解。新方法将是一个重大的改进，目前可用的方法来研究膜蛋白之间的关联。因此，该项目将为广泛的科学界提供更好的工具来研究膜蛋白的折叠、结构和功能。最终，方法的改进将导致发现阐明膜蛋白相互作用在细胞生命和人类疾病中的作用。此外，该计划将支持研究生的资助，并提高来自不同背景的本科生的教育经验。该项目由生物科学理事会分子与细胞生物科学部的分子生物物理学和数学与物理科学理事会化学部的实验物理化学项目共同支持。

Lateral interactions between hydrophobic transmembrane (TM) helices in cellular membranes underlie the folding of multi-span membrane proteins and signal transduction mediated by receptor tyrosine kinases (RTKs). Quantitative measurements of dimerization energetics in membranes, required for deciphering these processes, are challenging to perform due to many limitations of current experimental methodologies. The PI will develop novel versatile methodologies for studies of TM helix dimerization in biological membranes. The PI will carry out measurements in eukaryotic membranes and introduce a FRET-Western assay that probes both the stability and the structure of membrane proteins in the context of the native cellular membrane. The PI will also develop surface-supported lipid bilayers that mimic the plasma membrane by integrating asymmetry in lipid composition and uni-directional orientation of the proteins. This work will reveal the role of the complex plasma membrane environment in membrane protein interactions. The FRET-Western will allow the identification of mutations in TM domains that alter TM dimer stability or TM dimer structure, thus providing new insights into the structural determinants of protein dimerization in membranes. The new methods will be a significant improvement over the methodology that is currently available to study association between membrane proteins. Thus, the project will provide the broad scientific community with better tools to study membrane proteins folding, structure, and function. Ultimately, improvement in methods will lead to discoveries that elucidate the role of membrane protein interactions in cell life and human disease. Furthermore, this project will support the funding of graduate students and enhance the educational experience of undergraduate students from diverse backgrounds. This project is jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Experimental Physical Chemistry Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.