Structural Analysis of the ATP Synthase Membrane Domain

DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.