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.Includes bibliographical reference

Evolution of the electronic band structure of twisted bilayer graphene upon doping

The electronic band structure of twisted bilayer graphene develops van Hove singularities whose energy depends on the twist angle between the two layers. Using Raman spectroscopy, we monitor the evolution of the electronic band structure upon doping using the G peak area which is enhanced when the laser photon energy is resonant with the energy separation of the van Hove singularities. Upon charge doping, the Raman G peak area initially increases for twist angles larger than a critical angle and decreases for smaller angles. To explain this behavior with twist angle, the energy of separation of the van Hove singularities must decrease with increasing charge density demonstrating the ability to modify the electronic and optical properties of twisted bilayer graphene with doping.Comment: 10 pages, 4 figure

Single-molecule observation of nucleotide induced conformational changes in basal SecA-ATP hydrolysis

11 pages ; illustrationsSecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contribute to this motion during the transition state of the ATP hydrolysis cycle.Funding: This work was supported by the National Science Foundation (CAREER award number 1054832 to G.M.K.) and a Burroughs Wellcome Fund Career Award at the Scientific Interface (to G.M.K.)Nagaraju Chada1*, Kanokporn Chattrakun1, Brendan P. Marsh1†, Chunfeng Mao2, Priya Bariya2, Gavin M. King1,2‡: 1Department of Physics and Astronomy, University of Missouri–Columbia, Columbia, MO 65211, USA. 2Department of Biochemistry, University of Missouri–Columbia, Columbia, MO 65211, USA. *Present address: Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA. †Present address: Department of Applied Physics, Stanford University, Stanford, CA 94305 USA. ‡Corresponding author.Nagaraju Chada (1*), Kanokporn Chattrakun (1), Brendan P. Marsh (1†), Chunfeng Mao (2), Priya Bariya (2), Gavin M. King (1,2‡) -- References: 1) Department of Physics and Astronomy, University of Missouri–Columbia, Columbia,MO 65211, USA ; 2) Department of Biochemistry, University of Missouri–Columbia, Columbia, MO 65211, USA ; *) Present address: Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA ; †) Present address: Department of Applied Physics, Stanford University, Stanford, CA 94305 USA ; ‡) Corresponding author

Magnetic and structural properties of zinc-doped nickel ferrite

We have used dc-magnetization, ac-magnetic susceptibility and x-ray diffraction (XRD) to investigate the microscopic origin of the magnetic property modifications induced by zinc doping in bulk and nano-sized nickel ferrite. Magnetic measurements indicate that the nanoparticle energy barrier to magnetization reversal, EB, and the bulk saturation magnetization, M s, follow similar dependences on the zinc doping fraction, x. Both these magnetic quantities initially increase with increasing x, reach a maximum at x∼0.5, and eventually decrease upon further doping. Synchrotron and laboratory powder XRD data show no evidence of significant structural modifications for x∼0.5: we find the lattice parameter of ZnxNi1-xFe2O 4 to exhibit a linear increase with x, and the inverse spinel crystal structure of NiFe2O4 (x=0) to persist throughout the entire zinc doping range 0≤x≤1. Instead, A- and B- site occupancy refinements via Rietveld analysis reveal an indirect mechanism by which Ni2+ ions, which reside in B (octahedral) sites, are replaced by Zn2+. In this scenario, zinc is incorporated into A (tetrahedral) sites where it displaces Fe3+ ions, which migrate to the B sub-lattice and replace Ni2+. This doping mechanism is consistent with the observed magnetic property enhancement at x∼0.5