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Updated: Jul 23, 2025

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR
Published on: November 29, 2014
Time-Resolved Mn2+ -NO and NO-NO Distance Measurements Reveal That Catalytic Asymmetry Regulates Alternating Access
Michael Rudolph1, Robert Tampé2, Benesh Joseph1
1Department of Physics, Freie Universität Berlin, Arnimallee 14, 14195, Berlin, Germany.
Abstract:
ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO-NO, Mn2+ -NO, and Mn2+ -Mn2+ pulsed electron-electron double-resonance spectroscopy of TmrAB in a time-resolved manner. This type-IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn2+ -ATP, and Mn2+ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates the reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process.
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