Quantitation of Lipid-Protein Interactions and the Maintenance of Critical Infrastructure for Scientific Enterprise

Inwardly Rectifying K+ (Kir) channels are homotetrameric membrane proteins that are regulate the K+ flux into excitable cells. These channels are critical to establishing and setting the resting membrane potential that is critical for cellular electrical function in cells found in the brain, heart, kidneys, and skeletal muscle. As such their disfunction and mutation implicated in a myriad of disease conditions associated with these tissues including Parkinson’s, epilepsy, alcoholism, and cardiac arrhythmias. Thus, study of these channels remains of great importance. Like human Kir channels, KirBac1.1 is known to be regulated by membrane lipids; most notably in the case of KirBac1.1, activated by anionic phospholipids. In this dissertation, I apply solid-state NMR spectroscopy to characterize the lipid protein interactions of prokaryotic transmembrane potassium channel KirBac1.1. This works seeks to advance the understanding of the activation mechanism by characterizing the contacts between proteins and lipids. To date, structural elucidation of the open and active state of this channel remains elusive. To better understand these protein-lipid interactions, we investigate the lipid accessibility of KirBac1.1 Here, I apply a new method of lipid edited solid state NMR spectroscopy to ascertain site specific lipid contacts to the protein. This work experimentally corroborates a partially lipid mediated allosteric regulation pathway and sheds new light on residues involved in the regulation mechanism. Included additionally in this dissertation is a manual for performing basic Nuclear Magnetic Resonance facility maintenance. Historically, scientific instrumentation has been maintained and serviced in large part by in-house personnel, however in recent years, there has been a trend to simply pay for service contracts to have the instruments maintained. While this option is often convenient, the importance of having on-site staff with knowledge of repairs cannot be overstated. I have included directions for solving common problems, tips for keeping instruments in good working order, and other advice that preserves institutional knowledge gleaned from paid NMR service engineers, NMR researchers, and personal experience.