Structural studies on functional modules of ion-translocating ATPases

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ATP-synthases are large membrane protein complexes which are capable to convert reversibly the energy of electrochemical gradients into the chemical synthesis of ATP. The use of a crankshaft-like rotor, the g subunit, whose rotation inside the catalytic F1 portion couples to ATP-synthesis makes ATP-synthases the classical prototypes of 'molecular machines'. A concise understanding how energy coupling is performed by ATPsynthases still requires structural knowledge of its membraneembedded portion, the ion-conducting F0 sector, and of its attachment to the peripheral F1 portion. Soluble modules of a static stalk connector between the F0 and F1 portions of the Wolinella succinogenes ATP-synthase shall be overexpressed in E. coli, crystallized and analyzed by X-ray crystallography. Cocrystallization of stalk modules with the isolated F1 portion will reveal the mode of static attachment of the F1 portion to the membrane. Preparations of the intact proton-driven F0F1 synthase from W. succinogenes and its sodium-driven homologue from Ilyobacter tartaricus will be used in low-resolution studies with cryo-electron tomography and subjected to 3-D crystallization under conditions which arrest the ATP-synthases in a unique conformational mode. Putative lipid components which might play an important role for the assembly and stability of the F0 sector will be examined by nano ESI-MS methods.