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Updated: Jul 30, 2025

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis
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Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Published on: January 7, 2017

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

Daniel Mann1,2,3, Kristin Labudda1,4,5, Sophie Zimmermann1,4

  • 1Ruhr University Bochum, Department of Biophysics, Universitätsstraße 150, D-44780 Bochum, Germany.

Biological Chemistry
|May 15, 2023
PubMed

Insights

This study uses time-resolved FTIR spectroscopy to reveal the ATP binding and hydrolysis dynamics of Escherichia coli ATPase MsbA, a key multidrug resistant ABC transporter. We determined reaction rates and identified spectral signatures of key intermediates, advancing our understanding of lipid transport mechanisms.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Microbiology

Background:

  • Escherichia coli ATPase MsbA is a lipid flippase and a model for multidrug resistant ABC transporters.
  • Its physiological role involves transporting lipopolysaccharides for outer membrane construction in Gram-negative bacteria.
  • Previous studies lacked detailed insights into the dynamic coupling of ATP hydrolysis to transport.

Purpose of the Study:

  • To investigate the dynamic processes of ATP binding and hydrolysis in full-length MsbA.
  • To characterize the reaction kinetics and spectral signatures of MsbA's functional cycle.
  • To provide a foundation for future FTIR studies on MsbA-mediated transport.

Main Methods:

  • Time-resolved Fourier transform infrared (FTIR) spectroscopy was employed to monitor ATP binding and hydrolysis.
  • Kinetic rates were determined for wild-type MsbA and characterized using variants (MsbA-H537A, MsbA-S378A, MsbA-S482A) and photocaged nucleotides.
  • FTIR provided spectral signatures of reactants, products, and protein states.

Main Results:

  • Determined reaction rates for ATP binding (k1 = 0.49 ± 0.28 s⁻¹) and hydrolysis (k2 = 0.014 ± 0.003 s⁻¹) at 288 K.
  • Verified kinetic rates using photocaged nucleotides and a hydrolysis-deficient mutant (MsbA-H537A).
  • Observed that ADP remains protein-bound post-hydrolysis and identified spectral changes linked to disrupted hydrogen bonding in MsbA variants.

Conclusions:

  • FTIR spectroscopy is a powerful tool for dissecting the functional cycle of MsbA.
  • The study elucidates key steps in the ATP hydrolysis mechanism of this essential bacterial transporter.
  • Findings pave the way for advanced FTIR investigations into MsbA's allocrite transport mechanism.
Keywords:
ABC transporterATP hydrolysisFTIRintegral membrane protein

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