Your browser doesn't support javascript.
loading
ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy.
Mann, Daniel; Labudda, Kristin; Zimmermann, Sophie; Vocke, Kai Ulrich; Gasper, Raphael; Kötting, Carsten; Hofmann, Eckhard.
Afiliação
  • Mann D; Ruhr University Bochum, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany.
  • Labudda K; Forschungszentrum Jülich GmbH, Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons / ER-C-3: Structural Biology, D-52425 Jülich, Germany.
  • Zimmermann S; Forschungszentrum Jülich GmbH, Institute for Biological Information Processing / IBI-6 Cellular Structural Biology, D-52425 Jülich, Germany.
  • Vocke KU; Ruhr University Bochum, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany.
  • Gasper R; Ruhr University Bochum, Protein Crystallography, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany.
  • Kötting C; Ruhr University Bochum, Center for Protein Diagnostics (PRODI), Biospectroscopy, D-44780 Bochum, Germany.
  • Hofmann E; Ruhr University Bochum, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany.
Biol Chem ; 404(7): 727-737, 2023 06 27.
Article em En | MEDLINE | ID: mdl-37185095
ABSTRACT
The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k 1 = 0.49 ± 0.28 s-1 and k 2 = 0.014 ± 0.003 s-1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k 2 < 2 × 10-4 s-1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.
Assuntos
Palavras-chave

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Bactérias / Proteínas de Escherichia coli Idioma: En Revista: Biol Chem Assunto da revista: BIOQUIMICA Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Alemanha

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Bactérias / Proteínas de Escherichia coli Idioma: En Revista: Biol Chem Assunto da revista: BIOQUIMICA Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Alemanha