My overall scientific interests focus on the understanding of the correlation between the structure and function of membrane proteins.  Within the membrane lipid bilayer, membrane proteins exert a role in a vast array of cellular processes such as energy transduction, cell adhesion, ion channel conductance and cell signaling.  Membrane proteins are related to multidrug resistance, a hinder to cancer chemotherapy and to the action of antibiotics, and their malfunction is associated with cancer, genetic and infectious disorders.  In spite of their significance, less than one out of 500 high-resolution structures that are available belongs to membrane proteins.  Difficulties in the structural analysis, arise in part due to difficulties in obtaining adequate protein expression yields, difficulties in deriving significant structural information by conventional structural determination approaches, and/or limitations in achieving a suitable membrane-mimetic environment which preserve a native fold.   

We have previously studied the proton-translocating subunit c of F1Fo ATP synthase from an alkaliphic bacterium, a system which presents particular bioenergetic challenges.  An F1Fo ATP synthase, uses the transmembrane electrochemical gradient generated by electron transport through the respiratory chain to drive the chemical synthesis of ATP. During ATP synthesis, protons moving through the Fo membrane-spanning portion down the electrochemical gradient are believed to induce rotation of the complex’s rotor within Fo, causing long range conformational changes in the F1 catalytic portion. Revolution of the Fo-rotor in the opposite direction to that of ATP synthesis, triggers instead ATP hydrolysis. A proton/ATP stoichiometry of 3-4 protons transported per molecule of ATP synthesized or hydrolyzed is generally ascribed to the F1Fo ATP synthase.

Alkaliphilic bacteria on the other hand, accomplish optimal aerobic growth at an external pH of 10.5, while the cytoplasm remains at pH 8.3.  The difference in pH across the membrane (ΔpH), diminishes the proton motive force (Δp) which sustains ATP synthesis, while there is an insufficient rise in the transmembrane electrical potential (ΔΨ) to compensate.  Even at a submaximal proton motive force, oxidative phosphorylation measured by a phosphorylation potential (ΔGp), increases. An apparent contradiction also occurs in relation to Mitchell’s chemiosmotic model, as the imposition of artificial diffusion potentials fails to energize ATP synthesis above an external pH of 9.3.

We performed heteronuclear magnetic resonance spectroscopy studies on the subunit c at high pH, from the alkaliphile Bacillus pseudofirmus OF4 to provide an insight into the structural role of alkaliphilic-unique amino acid substitutions.  We found that these alkaliphilic-unique amino acid substitutions, assume various structural roles, such as the preservation of interhelical distances, facilitation of packing interactions between helices and/or monomers, establishment of physical contacts for effective rotation within Fo and F1, and modulation of the local structure and/or flexibility perhaps enhancing proton transfer. We have also generated a model of the alkaliphilic subunit a, that along with future OF4 c high-resolution structures, may contribute in the future to a structural-functional understanding of oxidative phosphorylation in alkaliphilic bacteria.