Biochemical and structural properties of yeast F1FO ATP synthase---Generation of an enzyme form amenable for crystallization

Project I. Membrane proteins, such as the F1F O complex, often exhibit detergent and phospholipid specific functional integrity. A detailed understanding of the stability of F1F O complex in different detergent solutions and the effect of phospholipids on functional integrity is of primary importance. In this thesis, detergents compatible for purification of yeast F1FO ATP synthase complex were identified by determining structural and functional stability of the complex in detergent containing solutions. Results indicate that the non-ionic class of detergents influence the behavior of purified complex to varying degrees with regard to solubility, structural integrity, and activity. In general, the purified F1FO complex in the nonionic detergent solutions, dodecylmaltoside, undecylthiomaltoside, ANAPOE-C 12E8 and nonylthioglucoside, are capable of showing high ATPase specific activity that is well coupled to proton pumping. Purified F1FO complex in detergent solutions also show strict dependence on externally added phospholipids to activate maximal ATPase specific activity and coupling of ATPase to proton pumping. The details of the detergent and phospholipid-specific behavior of the yeast F1FO complex led to (a) development of a robust in vitro assay to measure the structural and functional integrity of purified F1F O preparations, (b) design effective crystallization screens comprised of the effective detergents and lipids. The lipid and detergents identified are shown to be successful in crystallization trials of F1F O enzyme, and (c) a model explaining the role of lipids in the function of the F1FO ATP synthase.;Project II. We tested the hypothesis that increasing the size of the central stalk of yeast F1FO ATP synthase enzyme would sterically block the rotation of the central stalk, which forces the enzyme into a single rotomer conformation. A rotationally blocked enzyme form would be conformationally more homogeneous and therefore be more amenable for crystallization. Toward this end, C-terminal globular protein fusions of various sizes to the delta and epsilon subunits were tested for their ability to block the rotation. Results indicate that a 27 kDa Green fluorescent protein fused to the C-terminus of epsilon- subunit blocks the rotation of the enzyme in the direction of synthesis and hydrolysis. The 12 kDa and smaller fusions fail to show any significant effect on rotation. Steric blockage of rotation due to an increase in the size of central stalk also provides evidence for the rotational mechanism of the yeast ATPase complex.;Finally, the yeast strain expressing the rotationally blocked F 1FO complex was subjected to random mitochondrial mutagenesis and selected for enhanced expression of the GFP protein. Sequencing the mitochondrial DNA coding for the FO genes from the expression-enhanced strain showed a isoleucine to tyrosine mutation in the ATP9 gene indicating its significance in the enhanced expression.;Project III. The gamma-subunit in humans is regulated by an alternative splicing mechanism resulting in two tissue specific isoforms, the liver isoform and the heart isoform. The liver isoform of gamma has an extra aspartate at the highly conserved C-terminal end. Based on the careful regulation and high conservation the extra aspartate is proposed to have significance on the catalysis of the enzyme. A model developed from the F1 crystal structure shows the possibility of salt linkage between aspartate of the gamma-subunit and the arginine of the alphaE-subunit, which could affect the kinetics of the enzyme.;Overall, our results from the ATPase activity analysis and crystal structure suggests that the salt linkage is either not formed or if formed is not able to significantly alter the interaction of the C-terminus of gamma-subunit with the collar region. (Abstract shortened by UMI.)...