Mapping the ATP binding site in the plasma membrane h+-ATPase from Kluyveromyces lactis
Por:
Sampedro J.G., Nájera H., Uribe-Carvajal S., Ruiz-Granados Y.G.
Publicada:
1 nov 2014
Resumen:
The plasma membrane H+-ATPase from Kluyveromyces lactis contains 14 tryptophan residues. Binding a nucleotide or unfolding with Gnd-HCl quenched intrinsic fluorescence by?˜60 % suggesting that in the H+-ATPase-Nucleotide complex there is solvent-mediated collisional quenching of W505 fluorescence. N-bromosuccinimide (NBS) treatment of H+-ATPase modified a single W residue in both native and Gnd-HCl-unfolded H+-ATPase. Denaturing the H+-ATPase with 1 % SDS led to expose six tryptophan residues while requiring 17 NBS/H+-ATPase. The remaining eight tryptophan residues kept buried indicating a highly stable TM domain. Acrylamide generated static quenching of fluorescence; partial in the native enzyme (V?=?0.43 M-1) and complete in the Gnd-HCl-unfolded H+-ATPase (V?=?0.81 M-1). Collisional quenching (K sv) increased from 3.12 to 7.45 M-1 upon H+-ATPase unfolding. W505 fluorescence titration with NBS yielded a molar ratio of 6 NBS/H+-ATPase and quenched?˜?60 % fluorescence. In the recombinant N-domain, the distance between W505 and MantATP was estimated to be 21 Å by FRET. The amino acid residues involved in nucleotide binding were identified by N-domain molecular modelling and docking with ATP. In the N-domain/ATP complex model, the distance between W505 and ATP was 20.5 Å. ATP binding leads to a conformational change in the N-domain of H+-ATPase that exposes W505 to the environment. © 2014 Springer Science+Business Media New York.
Filiaciones:
Sampedro J.G.:
Departamento de Biofísica, Instituto de Física, Universidad Autónoma, San-Luis-Potosí-SLP, C.P. 78290, Mexico
Nájera H.:
Departamento de Ciencias Naturales, Universidad Autónoma Metropolitana Cuajimalpa (UAM-C), Pedro Antonio de los Santos 84, San-Miguel-Chapultepec, C.P. 11850 D.F, Mexico
Uribe-Carvajal S.:
Univ Nacl Autonoma Mexico, IFC, Dept Mol Genet, Mexico City 04510, DF, Mexico
Ruiz-Granados Y.G.:
Univ Nacl Autonoma Mexico, IFC, Dept Mol Genet, Mexico City 04510, DF, Mexico
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