UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
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Item NANOSCALE THERMODYNAMICS OF MECHANOSENSITIVE ION CHANNELS AND THEIR ROLE IN THE MECHANISM OF OSMOTIC FITNESS OF MICROBES(2018) Cetiner, Ugur; Sukharev, Sergei; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial mechanosensitive channels are major players in cells’ ability to cope with hypo-osmotic stress. Excess turgor pressure due to fast water influx is reduced as the channels, triggered by membrane tension, open and release osmolytes. In bacteria, the bulk release of ions and other osmolytes is mainly mediated by two families of mechanosensitive channels: MscS and MscL. The MscL family channels form large non-selective pathways in the membrane and gate near the lytic tension. In this way, they act as a final back up mechanism against osmotic downshock. MscS family channels require less tension to open and display great diversity in structure and functionality. Chapter 2 describes the first multifaceted phenomenological study of the emergency osmolyte release system in wild type Pseudomonas aeruginosa in comparison with E.coli. We recorded the kinetics of cell equilibration reported by light scattering responses to osmotic up- and down-shocks using the stopped-flow technique. We also performed the first electrophysiological characterization of the mechanosensitive ion channels in Pseudomonas aeruginosa. We presented a quantitative biophysical description of “osmotic fitness” which would be of interest to microbiologists, epidemiologists, ecologists and general environmental scientists. Chapter 3 presents the combined theoretical and experimental analysis of the full functional cycle of the bacterial channel MscS, which plays a major role in osmotic adjustments and environmental stability of most bacteria. We modeled MscS gating as a finite state continuous-time Markov chain and obtained analytical expressions for the steady state solution and the inactivated state area (which is experimentally hard to determine). In Chapter 4, we derived a general formalism to extract the free energy difference between the closed and open states of mechanosensitive ion channels (ΔF) from non-equilibrium work distributions associated with the channels’ gating. Our new approach bridges the gap between recent developments in non-equilibrium thermodynamics of small systems and ion channel biophysics. Our study also serves as an experimental verification of non-equilibrium work relations in a biological system. Therefore, the results in this thesis are sufficiently general and would be of interest to a broad community.Item THE GATING OF THE BACTERIAL MECHANOSENSITIVE CHANNEL MSCS REFLECTS ITS FUNCTION AS A SENSOR OF BOTH CROWDING AND LATERAL PRESSURE AS WELL AS ITS ROLE IN OSMOREGULATION(2014) Rowe, Ian Donald; Sukharev, Sergei I; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The mechanosensitive channel MscS is a ubiquitous bacterial membrane valve that opens by increased tension in the event of osmotic down-shock, releasing small internal osmolytes and thus preventing the cell from excessive hydration and possible lysis. This osmolyte release is accompanied by a reduction of osmotic pressure and volume of the cell, which simultaneously increases crowding. The large catalogue of MscS homologs in both prokaryotes and eukaryotes makes the study of this channel enticing to the field of physical biochemistry. Here are the results of three different studies, two of which focus on the gating of MscS in the presence of large osmolytes and amphipathic compounds and a third which describes the first electrophysiological examination of the inner membrane of the facultative pathogen Vibrio cholerae. The first study in Chapter 2 describes the sensitivity of gating transitions in MscS to large intracellular macromolecules. This sensitivity originates at the cytoplasmic cage domain and the perceived crowding alters the rate of opening, closing and inactivation. Chapter 3 details the utilization of MscS as a sensor for changes in the lateral pressure profile of native bilayers and how this technique can be used to resolve the potential of antibacterial agents to partition into the membrane. The third and final study describes our development of a procedure to generate giant spheroplasts of Vibrio cholerae and the subsequent characterization of its two major mechanosensitive channels in terms of gating, inactivation, conductivity, and compatible osmolyte sensitivity as well as the durability of the pathogen in response to osmotic shock. These contributions to the field of mechanobiology and channel biophysics suggest that environmental feedback during osmoregulation is recognized by the cell, provide a potential method to monitor the partitioning of antibiotics into a cell membrane, and lastly detail the mechano-electrical response of a relevant, disease-causing bacteria.Item The small mechanosensitive channel: Adaptive gating and timing during hypoosmotic shock.(2011) Boer, Miriam Sara; Sukharev, Sergei I.; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mechanosensation is the ability to respond to mechanical stimuli. It is present in many organisms. In Escherichia coli (E. coli), mechanosensation manifests in two membrane channels, the large mechanosensitive channel (MscL) and the small mechanosensitive channel (MscS). Both osmoregulatory channels sense membrane tension. MscS, the subject of these studies, consists of a transmembrane region and a cytoplasmic cage. It opens at tensions below those which can cause immediate damage to membranes, contrasting with MscL which opens at near-lytic tensions. Because it opens at non-threatening tensions, MscS not only opens and closes but also inactivates. Inactivation is non-conductive and tension insensitive, and this adaptive behavior was first observed on patch clamp. With the aid of carefully constructed molecular models, the first part of this dissertation evaluates whether inactivation is merely a patch clamp artifact or if it is indeed a part of in vivo MscS function. Working with wild type (WT) alongside fast inactivating and noninactivating mutants proved inactivation does confer a survival advantage to hypoosmotically shocked bacteria. Additionally, light scattering was used to view the swelling and channel response events in WT and knock out (KO) cells lacking mechanosensitive channels upon instantaneous hypoosmotic shock by way of stopped flow.