Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution.
Vma5p (subunit C) of the yeast V-ATPase was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that Vma5p comprises 64% alpha-helix and 17% beta-sheet content. The molecular mass of this subunit, determined by gel filtration analysis and small angle X-ray scattering (SAXS), was approximately 51+/-4 kDa, indicating a high hydration level of the protein in solution. The radius of gyration and the maximum size of Vma5p were determined to be 3.74+/-0.03 and 12.5+/-0.1 nm, respectively. Using two independent ab initio approaches, the first low-resolution shape of the protein was determined. Vma5p is an elongated boot-shaped particle consisting of two distinct domains. Co-reconstitution of Vma5p to V1 without C from Manduca sexta resulted in a V1-Vma5p hybrid complex and a 20% increase in ATPase hydrolysis activity.
Armbrüster
Andrea
A
Universität des Saarlandes, Fachrichtung 2.5 - Biophysik, Universitätsbau 76, D-66421 Homburg, Germany.
Svergun
Dmitri I
DI
Coskun
Unal
U
Juliano
Sandra
S
Bailer
Susanne M
SM
Grüber
Gerhard
G
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D018402:Manduca; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*; D014867:Water / Q000378:metabolism; D014965:X-Rays
D029701:Saccharomyces cerevisiae Proteins; C447616:VMA5 protein, S cerevisiae; D014867:Water; D004247:DNA; D025262:Vacuolar Proton-Translocating ATPases