Expression, purification and secondary structure analysis of Saccharomycescerevisiae vacuolar membrane H+-ATPase subunit F (Vma7p)

The vacuolar H+-ATPase is an acid pump found in virtually all eukaryotic ce lls. It shares a common macromolecular organization with the F1F0-ATPase, a nd some V-ATPase subunits are structural and functional homologues of F-ATP ase components. However, the vacuolar complex contains several subunits whi ch do not resemble F-ATPase subunits at the sequence level, and which curre ntly have no specific function assigned. One example is subunit F, the Vma7 p polypeptide of Saccharomyces cerevisiae. A recombinant form of Vma7p was expressed in Escherichia coli and purified to homogeneity. Mass spectroscop y confirmed a mass of 13 460 Da for Vma7p, and dynamic light scattering sho wed that the polypeptide was globular and monodisperse even at high concent rations. Analysis of secondary structure by circular dichroism and FTIR sho wed that Vma7p comprises 30% alpha -helix and 32-42% beta -sheet. The prote in fold recognition programme 'Threader 2' produced highly significant matc hes between Vma7p and five alpha-beta sandwich folds. Relative proportions of secondary structure elements within these folds were broadly consistent with the spectroscopic data. Although Vma7p does not share sequence similar ity with the F-ATPase epsilon subunit, the analysis suggests that the polyp eptides not only have similar masses and assemble into homologous core comp lexes, but also share similar secondary structures. It is possible that the two polypeptides are homologous and perform similar functions within their respective ATPases. The production of high yields of homogeneous, folded, monodisperse protein will facilitate high resolution crystallography and NM R spectroscopy studies.