Bridging biochemical activities with conformational dynamics observed in atomic force microscopy

This dissertation was written with the intention to provide future investigators with general information sufficient to start their own investigations in biological atomic force microscopy. It is noted that background information from both physics and biology has been included for overall clarity. A main emphasis of my thesis work was on modifying traditional assays to measure biochemical activities of membrane proteins adsorbed on surfaces prepared in an identical manner for atomic force microscopy (AFM) measurements. Additional projects included probing conformational dynamics of enzymes and utilizing atomic force spectroscopy to probe peptide-lipid interactions at enhanced temporal resolution via focused ion beam (FIB) modified AFM cantilevers. The experimental procedures in the appendix were purposefully written in a step by step format, with detailed notes of important or tricky aspects and precautions. Thus, these sections could serve as practical templates to construct future protocols and experiments. A chapter on future directions serves as suggestions of possible avenues of research. AFM measurements can shed light on membrane protein conformational dynamics and folding at a single molecule level. However, the unavoidable close proximity of the supporting surface to AFM specimens raises questions about the viability and preservation of biochemical activities. We quantified activities of the translocase from the general secretory (Sec) system of Escherichia coli, (E. coli), via two biochemical assays in surface supported bilayers: ATP hydrolysis and translocation. The ATP hydrolysis assays revealed distinct levels of activation ranging from low (basal), to medium (translocase-activated), to high (translocation-associated) corresponding to binding partners of SecA, the ATPase enzyme that hydrolyzes ATP. The measured on surface ATP hydrolysis activity levels were similar to traditional solution experiments. Furthermore, the surface activity assays uncovered characteristics of conformational hysteresis of SecA. Translocation assays displayed turn over numbers that were comparable to solution but with a reduction in the apparent rate constant. Despite a 10-fold difference in kinetics, the chemomechanical coupling (ATP hydrolyzed per residue translocated) only varied twofold on glass compared to solution. The activity changed with the topography of the supporting surface underneath the lipid bilayer. Glass cover slips have higher surface roughness than that of mica; this roughness can provide extra submembrane space. In turn, this extra space could lower the frictional coupling between the translocating polypeptide and the supporting surface. For these reasons, glass surfaces were favored over mica. Neutron reflectometry corroborated the results and provided characterization of the integral and peripheral components, as well as the submembrane space between the surface and the lower bilayer leaflet. Overall, surface activity assays had sufficient sensitivity to distinguish different levels of ATP hydrolysis and translocation activities of surface adsorbed systems, albeit with a slower rate-limiting step than observed in solution assays. Equipped with biochemical activity information for the surface-adsorbed proteins, we could then more strongly correlate conformational dynamics of the proteins observed in AFM measurements to their biochemical activities. We conducted AFM investigations on conformational dynamics of SecA on mica surfaces yielding fruitful information to specify the domain responsible for conformational dynamics during the ATP hydrolysis cycle. We also investigated the dynamics of translocase complexes engaging in translocation of precursor proteins across the membrane surface. These experiments brought to light previously underappreciated precursor species dependent conformational dynamics of the translocase.