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ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy

  • Daniel Mann ORCID logo , Kristin Labudda ORCID logo , Sophie Zimmermann , Kai Ulrich Vocke ORCID logo , Raphael Gasper ORCID logo , Carsten Kötting ORCID logo EMAIL logo and Eckhard Hofmann ORCID logo EMAIL logo
Published/Copyright: April 27, 2023
Biological Chemistry
From the journal Biological Chemistry Volume 404 Issue 7

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.

Keywords: ABC transporter; ATP hydrolysis; FTIR; integral membrane protein
Corresponding authors: Carsten Kötting, Ruhr University Bochum, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany; and Ruhr University Bochum, Center for Protein Diagnostics, Biospectroscopy, Universitätsstraße 150, D-44780 Bochum, Germany, E-mail: ; and Eckhard Hofmann, Ruhr University Bochum, Protein Crystallography, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany, E-mail:

Funding source: Deutsche Forschungsgemeinschaft

Award Identifier / Grant number: SFB642, TP A22 and TP A1

Acknowledgments

We would like to thank Meike Priehn, Fabian Zeipert, Vanessa Granitzka and Jenny Kleinmann who contributed to this project within their practical courses. We thank Prof. Dr. Klaus Gerwert for continuing support during the project.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: We acknowledge financial support from the Deutsche Forschungsgemeinschaft: CK and EH were funded withing the SFB642, TP A1 and TP A22, respectively, and within the Research Training Group GRK2341 “MiCon”. CK also acknowledges funding by grant 321722360.

  3. Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/hsz-2023-0122).

Received: 2023-02-05
Accepted: 2023-04-07
Published Online: 2023-04-27
Published in Print: 2023-06-27

© 2023 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Highlight: Membrane Proteins from Structure to Function
  3. The Rauischholzhausen Transport Colloquium: membrane proteins from structure to function
  4. Determination of membrane protein orientation upon liposomal reconstitution down to the single vesicle level
  5. Interaction of RTX toxins with the host cell plasma membrane
  6. Interactions of Na+/taurocholate cotransporting polypeptide with host cellular proteins upon hepatitis B and D virus infection: novel potential targets for antiviral therapy
  7. Mycobacterial type VII secretion systems
  8. Lipid exchange among electroneutral Sulfo-DIBMA nanodiscs is independent of ion concentration
  9. Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters
  10. ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy
https://doi.org/10.1515/hsz-2023-0122
Keywords for this article
ABC transporter; ATP hydrolysis; FTIR; integral membrane protein

Articles in the same Issue

  1. Frontmatter
  2. Highlight: Membrane Proteins from Structure to Function
  3. The Rauischholzhausen Transport Colloquium: membrane proteins from structure to function
  4. Determination of membrane protein orientation upon liposomal reconstitution down to the single vesicle level
  5. Interaction of RTX toxins with the host cell plasma membrane
  6. Interactions of Na+/taurocholate cotransporting polypeptide with host cellular proteins upon hepatitis B and D virus infection: novel potential targets for antiviral therapy
  7. Mycobacterial type VII secretion systems
  8. Lipid exchange among electroneutral Sulfo-DIBMA nanodiscs is independent of ion concentration
  9. Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters
  10. ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy
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