Exploring Protein Dynamics Through Paradigms of Chaperone Action and Structural Disorder

Abstract

Protein folding and dynamics shape all aspects of biology. Far from being rigid structures, proteins are intrinsically dynamic and undergo hierarchical motions at different time scales. Models help build frameworks to understand complex biological phenomena. In particular, the cross-disciplinary fields of protein folding, and dynamics have benefited immensely by employing model proteins to establish mechanistic paradigms and theoretical models that collectively shape our ways of thinking about the biophysics of proteins. In this thesis, I have used molecular chaperones and intrinsically disordered protein regions as models to investigate how structural dynamics regulate the biological activity of proteins. Molecular chaperones assist in protein folding and prevent protein aggregation. The first part of my thesis explores the mechanism of action of the small ATP-independent chaperone Spy on a topologically complex client, apoflavodoxin. Previous studies have established the dual functions of Spy as a “folding-while-bound” chaperone and as a “holdase” that prevents aggregation. My findings show that the molecular determinant of Spy’s mechanism of action is its affinity for unfolded conformations of the client. Weak affinity interactions with various folding states of the client enables it to fold to its native state while being continuously held by Spy. However, when Spy binds too tightly to (partially) unfolded states, it acts as a kinetic trap that inhibits folding-while-bound. The second part of my thesis involves characterizing the structure and dynamics of the disordered protein SERF in the context of its RNA-binding properties. In my research, I investigate the interplay between the structural dynamics of SERF and its interaction with RNA, both in dilute solution and within phase-separated RNA-protein condensates. I have developed and characterized an in vitro model RNA-protein complex comprising SERF and the model helix-junction-helix motif found in the 5’ end of the human immunodeficiency virus type 1 (HIV-1) transactivating response region (TAR) RNA. The conserved N-terminal segment of SERF is disordered and interacts with RNA. Upon binding TAR, SERF undergoes global compaction, leading to slower structural dynamics. SERF and TAR can form condensates by associative phase separation. In the future, we will use the SERF-TAR system to study molecular features that drive the formation of RNA-protein condensates. Collectively, using two disparate systems offers insights into how the dynamically accessible conformational space of proteins determines their biological functions.