For an organism to survive, its proteins must adapt to respond to various challenges it faces in a living cell. One of these challenges is macromolecular crowding that can derail even robust biological pathways. This situation becomes critical when considering proteins with energetic folding landscapes that permit many conformational states. In these cases, the environment can clearly influence the conformation by favoring one pathway over another. Such decisions can have long-lasting biological consequences, as is the case for prions, proteins with the ability to exist stably in distinct functional states with divergent structures: one soluble, the other a highly ordered, self-templating aggregate. Because the aggregating proteins have identical sequences, differences in cellular environment are responsible for the conformational switch. Despite the importance of the environment for protein folding, structural investigations of biomolecules are typically confined to in vitro systems, which cannot capture important structural features imposed by biological environments. Solid-state NMR spectroscopy (ssNMR) is currently undergoing a 'sensitivity renaissance' with the development of dynamic nuclear polarization (DNP). This method has a theoretical maximum sensitivity enhancement of 660-fold; thus an experiment that would require decades of experimental time with traditional ssNMR methods can be collected in a day with DNP NMR. This project will advance our knowledge about protein structure in natural biological contexts. The PI will develop training videos for undergraduate and high school students in collaboration with high school teachers. These training videos will then be distributed nationally through the American Society for Biochemistry and Molecular Biology.
Structural investigations of biomolecules are typically confined to in vitro systems under extremely limited conditions. These investigations yield invaluable insights, but such experiments cannot capture important structural features imposed by cellular environments. Structural studies of proteins in their native contexts are not only possible using state-of-the-art sensitivity-enhanced (dynamic nuclear polarization or DNP) solid-state nuclear magnetic resonance (NMR) techniques, but also that the native context can have a dramatic influence on protein structure. This project will 1) visualize such structural changes with atomic level resolution and 2) provide an understanding of how genetic background can influence protein folding. To do so, the PI will use novel sample preparation for NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region. Using a self-polymerizing yeast prion protein, Sup35, this project will investigate how native and mutant cellular environments affect amyloid structure. While the methodology developed in this project can be easily applied to systems of interest such as the protein mis-folding in neurodegenerative diseases like Alzheimer's it can likewise be applied to emerging basic problems such as the structural basis of phase separated biological condensates. The work will build the foundation of a new field of in vivo structural biology directly informed by genetic backgrounds, environmental perturbation and cellular phenotypes.
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.
