Quantitative structural and functional studies of membrane transporter evolution

This research will investigate membrane protein evolution with the long-term goal of designing artificial cells with tailored functions. The specific objective of this project is to determine how mutations in membrane protein transporters impact dynamics, conformational equilibria, and the direction of transport, which will provide insight into the biogenesis of membrane proteins. This research will involve and train students and postdoctoral associates in biochemistry and biophysical chemistry. Professional training opportunities will be afforded to strengthen communication skills and to prepare trainees for future career paths. The project will also involve the development of a class for graduate students with the goal of teaching students advanced techniques in biochemistry and biophysical chemistry. The broader goal of this endeavor is to strike to a more equitable balance between lecture-based curricula and hands-on learning in graduate education. Many membrane protein transporters are comprised of a single polypeptide chain that contains inverted repeats within its structure. This research will investigate the underpinnings of membrane protein evolution by using mutations to derive a quantitative relationship between function and the free energy distinguishing conformational states required for function. The functional experiments will utilize a flow cytometry-based method capable of analyzing a library of mutations in a high throughput manner. To correlate the free energy with function, a quantitative method involving 19F NMR spectroscopy will be developed. Finally, theories about the evolution of ion-coupled transport mechanisms will be tested by using directed evolution experiments and bioinformatics. This project is supported by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Sciences. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.