Structural and dynamic studies of protein and protein-nucleic acid assemblies in health and disease

PROJECT SUMMARY Large protein and protein-nucleic acid complexes and assemblies play key roles in human health and disease, and understanding their three-dimensional structures, conformational dynamics and interactions at the atomistic level is essential for understanding their function and associated biological mechanisms. High-resolution structural and dynamic studies of such large biomacromolecular systems have generally come with significant challenges due to their inherently complex nature. However, by integrating the most recent advances in experimental and computational methodologies many of these obstacles can now be overcome. We develop and apply advanced spectroscopic and microscopic techniques, most notably multidimensional magic-angle spinning solid-state nuclear magnetic resonance and cryo-electron microscopy, in combination with complementary biochemical, biophysical and computational approaches to provide atomic-resolution insights into the structure, dynamics, interactions and function of large protein and protein-DNA systems of major significance in biology. The systems of primary interest in our studies include: (i) nucleosomes and large nucleosome arrays mimicking chromatin, a supramolecular complex of DNA with histone proteins that regulates essential genome functions such as transcription, replication and repair through dynamic changes in its structure, and their complexes with chromatin-binding proteins and (ii) filamentous amyloids formed by mammalian Y145Stop prion protein variants that exhibit strain and cross-seeding barrier phenomena akin to those that underlie the pathogenesis of prion and other neurodegenerative diseases. For chromatin and its complexes we aim to provide quantitative information on functionally-relevant conformational dynamics of histone tail and globular domains as a function of different chromatin parameters including nucleosome positioning and post- translational modifications, which modulate nucleosome plasticity and gene activity, and histone protein interactions with two representative chromatin-binding proteins including a histone reader corresponding to the histone acetyltransferase and ZZ-type zinc finger domains of human p300 and a yeast pioneer transcription factor Cbf1. Combined, these studies will advance the atomic level understanding of critical events that regulate gene transcription. For mammalian Y145Stop prion protein amyloids our studies will decipher the structural basis of amyloid strains and species- and strain-dependent cross-seeding barriers with atomic level detail and furnish insights into the impact of specific Gerstmann-Straussler-Scheinker disease linked amino acid mutations on the core structure of Y145Stop prion protein amyloid fibrils. Collectively, the insights provided by these studies have significant implications for the understanding of prion strains and seeding barriers, prion conformational switching and prion strain selection, which are all of fundamental importance in the pathogenesis of prion and other neurodegenerative disorders.