NSF/MCB-BSF The virtues of lanthanides and fluorine for tracking in-cell protein conformation: a marriage of NMR and EPR

The goal of this project is to develop experimental tools for understanding the structures of proteins inside living cells while the proteins carry out their functions. The tools to be developed will measure distances within and between proteins using a combination of in-cell nuclear magnetic resonance (NMR) spectroscopy and electron paramagnetic resonance (EPR) spectroscopy, two powerful approaches for tracking the structure and movement of a protein.

Development of these approaches for making measurements inside live cells will have broad application as we expect to obtain 1) benchmark data for in-cell NMR and EPR that can be used by other practitioners of these methodologies (biochemists, biophysicist) and 2) biological insight that may open new strategies for understanding protein function in cells and when bound by drugs. As such, the proposed research will impact several areas of science and engineering, including physical chemistry, structural biochemistry, magnetic resonance spectroscopy, biotechnology, and pharmacology.

The proposed innovative research program will provide unique training for students at all levels in state-of-the-art experimental methods. Importantly, a program within this project will empower emerging female scientists in magnetic resonance and provide a framework for populating the next generation of females in leadership roles in the global STEM workforce.

Biophysical analyses of biomolecular structure and properties is typically performed on the isolated biomolecule, removed from its native environment, such as the cell. By necessity, such analyses ignore or grossly simplify cellular influences. Although this strategy has provided and continues to provide indispensable information on molecular structure, it leaves open many questions about how that information relates to interactions and function within the cell, where innumerable organelles and molecular machines engage in an intricate dance, spatially and temporally, interacting and changing shape as they perform their tasks.

The main objective of this project is to begin addressing these open questions by developing an integrated 19F NMR/EPR approach that informs on the structures of proteins in their native cellular environments. Specifically, the program will establish a combined 19F-PRE (paramagnetic relaxation enhancement) and 19F-PCS (pseudo contact shift) NMR approach to measure distances in the range of 5-50 Å and a combined Gd(III)-Gd(III) DEER (double electron-electron resonance) and Gd(III)-19F ENDOR/ED-NMR (electron nuclear double resonance/electron-electron double resonance (ELDOR)-detected NMR) approach to measure distances in the 5-80 Å range.

Together, the united methodology will provide complementary information that is inaccessible when using either approach alone and will permit a more complete characterization of structure and intra- or intermolecular interactions in the context of function. This project is funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.

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.