Investigating Structural Dynamics of Periplasmic Proteins by NMR Spectroscopy

Abstract

Cell viability is constantly challenged by the presence of misfolded or aggregated proteins, which can disrupt protein homeostasis and threaten the cellular survival. To counter this, cells rely on a finely tuned system of molecular chaperones and proteases that continuously surveil and preserve the proteome integrity. Cellular protein quality control (PQC) operates through three main strategies whereby misfolded proteins are refolded, degraded, or sequestered within specialized compartments. Although present in all living organisms, PQC differs between eukaryotic and prokaryotic, with eukaryotic cells adopting a more complex and organized multi-layered PQC network, while prokaryotic cells are more prone to fast response to sudden stresses through simple but effective pathways. In particular, Gram-negative bacteria have developed a layered compartment, the periplasm, to protect the cell from external stressful conditions. In the periplasm, different proteins are involved in the periplasmic PQC and the periplasmic chaperone SurA plays a crucial role maintaining the proteostasis in outer membrane proteins (OMPs) biogenesis. After being transported across the inner membrane via the SecYEG complex, OMPs need to be transported to the b-barrel assembly machine (BAM) complex, which is responsible to correctly fold them into the outer membrane. In this delivery process, SurA acts as a holding shuttle that dynamically and transiently binds OMPs maintain them in an unfolded but still foldable state. Despite being the main periplasmic chaperone for most OMPs, SurA binding and delivering modes are still unclear. In this thesis, I focused on uncovering SurA dynamics as well as characterizing its interaction with its natural substrate OmpX by using solution nuclear magnetic resonance (NMR) spectroscopy as main method of study. I analyzed the backbone and methyl dynamics for wild-type SurA and compared them with the previously characterized mutant S220A, known to have a detached PPIase1 resulting in an open state which exposes SurA binding sites. This comparison between the closed (wild type) and open (S220A mutant) state of SurA allowed me to study SurA interaction with OmpX.