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.
Twenty to 30% of the open reading frames in organisms encode membrane proteins. Despite their abundance and key roles in cell signaling, cell adhesion, recognition, motility, energy production, and transport of nutrients and cholesterol, our knowledge of the folding and the structure-function relationship for membrane proteins is limited, and lags far behind that of soluble proteins. In part, this is due to limited biophysical tools to adequately probe the physical-chemical principles underlying membrane protein folding and structure. One of the simplest, yet critically important biological interactions is the lateral association of TM helices in cellular membranes. Such lateral interactions underlie the folding of multi-span membrane proteins with unique three dimensional structures, carrying out complex biochemical tasks. Quantitative measurements of dimerization energetics in membranes, required for deciphering these processes, are challenging to perform due to many limitations of current experimental methodologies. With the support of the NSF, our laboratory has developed novel versatile methodologies for studies of membrane protein interactions. We have carried out the first measurements of membrane protein dimerization in eukaryotic membranes. We have also introduced assays that probe both the stability and the structure of membrane proteins in the context of the native cellular membrane. These assays have allowed the identification of mutations in membrane proteins that alter dimer structure, thus providing new insights into the structural determinants of protein dimerization in membranes. We have also developed surface-supported lipid bilayers of varying lipid composition, to be used as models of biological membranes in biophysical studies. The methods developed under this award do not utilize specialized equipment and can be introduced in any membrane biophysics laboratory. These methods are a significant improvement over the methodology that is currently available to study association between membrane proteins. Thus, the proposed method development has provided the broad scientific community with better tools to study membrane proteins folding, structure, and function. Four graduate students working on this project have earned their Ph. Ds and have participated in the development of cutting edge new methodologies. They have worked together with undergraduate and high school students, enhancing their educational experience.
