The boxing glove shape of subunit d of the yeast V-ATPase in solution and the importance of disulfide formation for folding of this protein.
The low resolution structure of subunit d (Vma6p) of the Saccharomyces cerevisiae V-ATPase was determined from solution X-ray scattering data. The protein is a boxing glove-shaped molecule consisting of two distinct domains, with a width of about 6.5 nm and 3.5 nm, respectively. To understand the importance of the N- and C-termini inside the protein, four truncated forms of subunit d (d (11-345), d (38-345), d (1-328) and d (1-298)) and mutant subunit d, with a substitution of Cys329 against Ser, were expressed, and only d (11-345), containing all six cysteine residues was soluble. The structural properties of d depends strongly on the presence of a disulfide bond. Changes in response to disulfide formation have been studied by fluorescence- and CD spectroscopy, and biochemical approaches. Cysteins, involved in disulfide bridges, were analyzed by MALDI-TOF mass spectrometry. Finally, the solution structure of subunit d will be discussed in terms of the topological arrangement of the V(1)V(O) ATPase.
Thaker
Youg R
YR
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
Roessle
Manfred
M
Grüber
Gerhard
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D014961:X-Ray Diffraction
D017931:DNA Primers; D004271:DNA, Fungal; D004220:Disulfides; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C091579:VMA8 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases