Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution
Research output: Contribution to journal › Letter › Contributed › peer-review
Contributors
Abstract
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% α-helix and 17% β-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.
Details
Original language | English |
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Pages (from-to) | 119-125 |
Number of pages | 7 |
Journal | FEBS letters |
Volume | 570 |
Issue number | 1-3 |
Publication status | Published - 16 Jul 2004 |
Peer-reviewed | Yes |
Externally published | Yes |
External IDs
WOS | 000222885400023 |
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Scopus | 3142574553 |
PubMed | 15251451 |
Keywords
Keywords
- BSA, bovine serum albumin, IPTG, isopropyl-β-D-thio-galactoside, NTA, nitrilotriacetic acid, PAGE, polyacrylamide gel electrophoresis, PCR, polymerase chain reaction, SDS, sodium dodecyl sulfate, Tris, Tris-(hydroxymethyl) aminomethane