Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution

Andrea Armbrüster; Dmitri I. Svergun; Ünal Coskun; Sandra Juliano; Susanne M. Bailer; Gerhard Grüber

doi:10.1016/j.febslet.2004.06.029

Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution

Andrea Armbrüster, Dmitri I. Svergun, Ünal Coskun, Sandra Juliano, Susanne M. Bailer, Gerhard Grüber^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

42 Citations (Scopus)

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.

Original language	English
Pages (from-to)	119-125
Number of pages	7
Journal	FEBS Letters
Volume	570
Issue number	1-3
DOIs	https://doi.org/10.1016/j.febslet.2004.06.029
Publication status	Published - Jul 16 2004
Externally published	Yes

ASJC Scopus Subject Areas

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

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

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10.1016/j.febslet.2004.06.029

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title = "Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution",

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.",

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author = "Andrea Armbr{\"u}ster and Svergun, \{Dmitri I.\} and {\"U}nal Coskun and Sandra Juliano and Bailer, \{Susanne M.\} and Gerhard Gr{\"u}ber",

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AU - Armbrüster, Andrea

AU - Svergun, Dmitri I.

AU - Coskun, Ünal

AU - Juliano, Sandra

AU - Bailer, Susanne M.

AU - Grüber, Gerhard

PY - 2004/7/16

Y1 - 2004/7/16

N2 - 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.

AB - 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.

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KW - IPTG, isopropyl-β-D-thio-galactoside

KW - NTA, nitrilotriacetic acid

KW - PAGE, polyacrylamide gel electrophoresis

KW - PCR, polymerase chain reaction

KW - SDS, sodium dodecyl sulfate

KW - Tris, Tris-(hydroxymethyl) aminomethane

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Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution

Abstract

ASJC Scopus Subject Areas

Keywords

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