46 research outputs found

    ATP synthase: from single molecule to human bioenergetics

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    ATP synthase (FoF1) consists of an ATP-driven motor (F1) and a H+-driven motor (Fo), which rotate in opposite directions. FoF1 reconstituted into a lipid membrane is capable of ATP synthesis driven by H+ flux. As the basic structures of F1 (α3β3γδε) and Fo (ab2c10) are ubiquitous, stable thermophilic FoF1 (TFoF1) has been used to elucidate molecular mechanisms, while human F1Fo (HF1Fo) has been used to study biomedical significance. Among F1s, only thermophilic F1 (TF1) can be analyzed simultaneously by reconstitution, crystallography, mutagenesis and nanotechnology for torque-driven ATP synthesis using elastic coupling mechanisms. In contrast to the single operon of TFoF1, HFoF1 is encoded by both nuclear DNA with introns and mitochondrial DNA. The regulatory mechanism, tissue specificity and physiopathology of HFoF1 were elucidated by proteomics, RNA interference, cytoplasts and transgenic mice. The ATP synthesized daily by HFoF1 is in the order of tens of kilograms, and is primarily controlled by the brain in response to fluctuations in activity

    Estimating the Rotation Rate in the Vacuolar Proton-ATPase in Native Yeast Vacuolar Membranes

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    The rate of rotation of the rotor of the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or the steady parts of enzyme, is estimated in native vacuolar membrane vesicles of Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are spontaneously formed after exposing purified yeast vacuoles to osmotic shock. The fraction of the total ATPase activity originating from V-ATPase is determined using the potent and specific inhibi-tor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during 10 min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit to the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data is incompatible with models assuming more binding sites) to the inhibitor titration curve determines the concentration of the enzyme. Combining it with the known rotation:ATP stoichiometry of V-ATPase and the assayed concentration of inorganic phosphate liberated by V-ATPase leads to an average rate of ~9.53 Hz of the 360 degrees rotation, which, according to the time-dependence of the activity, extrapolates to ~14.14 Hz for the beginning of the reaction. These are low limit estimates. To our knowledge this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and it is not fixed on a solid support, instead it is functioning in its native membrane environment

    Single Molecule Biophysics - II

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    Protein Conformational Studies by Hydrogen/Deuterium Exchange Mass Spectrometry

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    Proteins are biological macromolecules responsible for the majority of all physiological processes. In order to properly function proteins are required to adopt highly ordered structures. These structural aspects may be found within a single protein or arise from multi-protein complexes. Here hydrogen/deuterium exchange mass spectrometry (HDX-MS) is employed as a tool to determine the extent of protein higher order structure. Exposure to D2O-based solvent causes the heavier isotope to exchange with amide hydrogens in the polypeptide backbone. This exchange is mainly dependent on protein conformation because the presence of stable hydrogen-bonded secondary structure will impede the incorporation of deuterium when compared to regions that are unstructured. In this work HDX-MS is used to study denaturant-induced unfolding of oxidized and reduced cytochrome c as well as ATP binding to the ε subunit of FOF1-ATP synthase. This work also lays the foundation to use this technique to study larger, more complex systems

    Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism

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    The ubiquitous biological nanomotors were classified into two categories in the past: linear and rotation motors. In 2013, a third type of biomotor, revolution without rotation (http://rnanano.osu.edu/movie.html), was discovered and found to be widespread among bacteria, eukaryotic viruses, and double-stranded DNA (dsDNA) bacteriophages. This review focuses on recent findings about various aspects of motors, including chirality, stoichiometry, channel size, entropy, conformational change, and energy usage rate, in a variety of well-studied motors, including FoF1 ATPase, helicases, viral dsDNA-packaging motors, bacterial chromosome translocases, myosin, kinesin, and dynein. In particular, dsDNA translocases are used to illustrate how these features relate to the motion mechanism and how nature elegantly evolved a revolution mechanism to avoid coiling and tangling during lengthy dsDNA genome transportation in cell division. Motor chirality and channel size are two factors that distinguish rotation motors from revolution motors. Rotation motors use right-handed channels to drive the right-handed dsDNA, similar to the way a nut drives the bolt with threads in same orientation; revolution motors use left-handed motor channels to revolve the right-handed dsDNA. Rotation motors use small channels (\u3c 2 nm in diameter) for the close contact of the channel wall with single-stranded DNA (ssDNA) or the 2-nm dsDNA bolt; revolution motors use larger channels (\u3e 3 nm) with room for the bolt to revolve. Binding and hydrolysis of ATP are linked to different conformational entropy changes in the motor that lead to altered affinity for the substrate and allow work to be done, for example, helicase unwinding of DNA or translocase directional movement of DNA

    Complementary Mass Spectrometry Methods for Characterizing Protein Folding, Structure, and Dynamics

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    Proteins are involved in virtually every biochemical process. A comprehensive characterization of factors that govern protein function is essential for understanding the biomedical aspects of human health. This dissertation aims to develop complementary mass spectrometry-based methods and apply them to solve problems pertaining to the area of protein structure, folding and dynamics. ‎Chapter 1 uses fast photochemical oxidation of proteins (FPOP) to characterize partially disordered conformers populated under semi-denaturing conditions. In FPOP, ·OH generated by laser photolysis of H2O2 introduces oxidative modifications at solvent accessible side chains. By contrast, buried sites are protected from radical attack. Using apomyoglobin (aMb), it was demonstrated that under optimized conditions undesired can be almost completely eliminated and detailed structural information can be obtained. ‎Chapter 3 combines FPOP with submillisecond mixing to enable studying early events in protein folding. aMb served as a model system for these measurements. Spatially-resolved changes in solvent accessibility follow the folding process. Data revealed that early aMb folding events are driven by both local and sequence-remote docking of hydrophobic side chains. Assembly of a partially formed scaffold after 0.2 ms is followed by stepwise consolidation that ultimately yields the native state. The submillisecond mixer used improved the time resolution by a factor of 50 compared to earlier FPOP experiments. Submillisecond mixing in conjunction with slower mixing techniques help monitor completes folding pathways, from fractions of a millisecond all the way to minutes. ‎Chapter 4 uses ion mobility mass spectrometry (IM-MS) to explore the structural relationship between semi folded solution and gas phase protein conformers. Collision cross sections (CCSs) provide a measure of analyte size. Mb was used as model system because it follows a sequential unfolding pathway that comprises two partially disordered states. IM-MS data showed that the degree of gas phase unfolding is not strongly correlated with the corresponding solution. Gas phase unfolding as well as collapse events can lead to disparities between gaseous and solution structures for partially unfolded proteins. IM-MS data on non-native conformers should therefore be interpreted with caution. ‎Chapter 5 uses HDX-MS to examine the role of conformational dynamics for the function of multi-protein molecular machines such as FoF1 ATP synthase. HDX-MS monitors backbone deuteration kinetics in the presence of D2O. Disordered segments exchange more rapidly than those in tightly folded regions. Measurements of spatially-resolved deuterium are performed using LC-MS. It was found that the H-bonding network of key power transmission elements is insensitive to PMF-induced mechanical stress. Unexpectedly, HDX-MS reveals a pronounced destabilization of the g C-terminus during rotational catalysis under PMF. The behavior of g is attributed to kinetic friction within the apical rotor bearing

    The unique histidine of F-ATP synthase subunit OSCP mediates regulation of the permeability transition by matrix pH

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    The “Permeability transition” (PT) is one of the most studied events that may trigger cell death and is due to a Ca2+- and ROS-dependent opening of a nonspecific pore, called PTP, whose molecular nature has been long debated. Recently, our research group has demonstrated that PTP forms from FoF1 ATP synthase dimers, demonstrating the ability of this complex to switch from the key enzyme for the aerobic synthesis of ATP into a potential cell death mediator. The goal of this PhD thesis has been to define which structural changes of ATP synthase are responsible for the pH modulation of PTP. Indeed, it is well known from nineties that the optimum matrix pH for PT occurrence is about 7.3, and a decrease leads to decreased probability of PTP opening. The pH effect has been ascribed to reversible protonation of His residues located on the PTP that can be blocked by the histidine modifying reagent diethyl pyrocarbonate (DPC). Moreover, in mammalian cells, similarly to the drug Cyclosporin A (CsA), acidic pH also promotes release from the inner membrane of the matrix protein Cyclophilin D (CyPD), which is a well-known PTP activator. As our group demonstrated that CyPD binds to the ATP Synthase OSCP subunit, mainly through electrostatic interactions and resulting in partial enzyme inhibition, the hypothesis has been advanced that the unique histidine located on OSCP, His112 according to bovine numbering, may be responsible for both the pH effects on CyPD (un)binding to ATP synthase and on PTP/ATP synthase opening. OSCPHis112 is exposed to the solvent and is located in the flexible linker region between the structured N- and C-terminal domains of OSCP. The results obtained by ATP synthase immunoprecipitation from bovine heart mitochondria showed that acidic pH induces CyPD release that is prevented by DPC, perfectly matching the effect of DPC on CyPD-PTP interaction. DPC also prevented the binding at low pH of the inhibitor protein IF1 to ATP synthase, but this effect is probably not relevant to PTP modulation. ESI-MS and ESI-MS/MS analyses of the OSCP isolated from DPC-treated mitochondria revealed that the 95-113 peptide shows a mass shift of +72 Da, which is indicative of carbethoxylation of the unique His112. These data therefore strongly support the hypothesis that OSCP His112 is part of the binding site of CyPD on the protein, so that its protonation by lowering pH favors CyPD release. Of note, this region contains several residues of glutamic acid conferring a low potential surface, which is complementary to the mainly high potential surface of CyPD. Consistently to this model, DPC inhibits the ATPase activity of ATP synthase only when CyPD is released from OSCP, i.e. in the presence of CsA and in mitochondria from CyPD-null mice. Replacement of OSCPHis112 with a Gln in HEK cells, by the CRISPR/Cas9 system, showed its involvement even in the effect of low pH on PTP opening. Indeed, the PTP open probability is not affected by acidic pH only in mutated cells, while DPC reverts the pH inhibition exclusively in wild type cells. Finally, evaluation of the structural stability of the ATP Synthase dimers at low pH by Blue-native PAGE excluded their destabilization, which could affect PTP formation. In summary, these data provide a convincing model for the pH modulation of PTP, as well as a compelling evidence that ATP synthase and PTP are the same molecular entity
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