The long term goal of this project is to understand how the catalytic reaction of the hydrolysis or synthesis of adenosine triphosphate is coupled to the transport of protons across the membrane in the multi-subunit FOF 1 ATP synthase. Recent observations have established that the catalytic and transport functions utilize rotational mechanisms and energy is transferred between them, in part, by the torque generated on the rotor subunits. Characterization of several mutant enzymes with substitutions in the interfaces between rotor and stator indicate that the efficiency of energy transfer between transport and catalysis involves transmission of conformational information. To understand how the conformational effects modulate the catalytic and transport mechanisms, we will carry out two Specific Aims.
Specific Aim 1 : the site-directed spin labeling strategy of EPR spectroscopy and determination of solvent accessibility of cysteine substitutions by reactivity with hydrophilic sulfhydryl reagents will be used to generate a structure-function map of the gamma and epsilon rotor subunits. Physical properties of the rotor and how it interacts with the stator will be elucidated.
Specific Aim 2 : we will dissect the coordination between the catalytic transition state and the rotational behavior of the catalytic motor. The effects of amino acid replacements on ATP-driven gamma subunit rotation, which is observed directly on a nano-fabricated experimental platform, will be correlated to the partial reaction steps of the catalytic mechanism, which are determined chemically by pre-steady state quench-flow and fluorescence stopped-flow kinetic measurements. Mutant enzymes will be analyzed that have amino acid replacements that perturb rotor-stator interactions and have effects on the catalytic transition state and rotation. These studies seek to understand the molecular mechanisms of a true molecular rotary motor, and in particular, the mechanisms of coupling efficiency that generate high torque forces which makes it the most powerful nanoelectromechanical motor.
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