Expression, purification and secondary structure analysis of Saccharomyces cerevisiae vacuolar membrane H+-ATPase subunit F (Vma7p).
The vacuolar H+-ATPase is an acid pump found in virtually all eukaryotic cells. It shares a common macromolecular organization with the F1F0-ATPase, and some V-ATPase subunits are structural and functional homologues of F-ATPase components. However, the vacuolar complex contains several subunits which do not resemble F-ATPase subunits at the sequence level, and which currently have no specific function assigned. One example is subunit F, the Vma7p polypeptide of Saccharomyces cerevisiae. A recombinant form of Vma7p was expressed in Escherichia coli and purified to homogeneity. Mass spectroscopy confirmed a mass of 13460 Da for Vma7p, and dynamic light scattering showed that the polypeptide was globular and monodisperse even at high concentrations. Analysis of secondary structure by circular dichroism and FTIR showed that Vma7p comprises 30% alpha-helix and 32-42% beta-sheet. The protein fold recognition programme 'Threader 2' produced highly significant matches 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 similarity with the F-ATPase epsilon subunit, the analysis suggests that the polypeptides not only have similar masses and assemble into homologous core complexes, 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 NMR spectroscopy studies.
Jones
R P
RP
School of Biochemistry and Molecular Biology, University of Leeds, UK.
Hunt
I E
IE
Jaeger
J
J
Ward
A
A
O'Reilly
J
J
Barratt
E A
EA
Findlay
J B
JB
Harrison
M A
MA
D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D008970:Molecular Weight; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C089600:VMA7 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases