A new approach based on nanofabricated zero-mode waveguides for observing stepwise binding in multimeric proteins at physiologically relevant concentrations with single-molecule resolution is demonstrated.
Significance and Impact
The approach overcomes the current limitations of ensemble measurements and offers several key insights into the mechanism of ligand activation.
– Cyclic adenosine monophosphate (cAMP) binding to purified pacemaker ion channels was quantified at the single-molecule level using zero-mode waveguides (ZMWs) to monitor binding.
– ZMWs enabled measurements at physiologically relevant protein concentrations for extended times providing an unprecedented view of early binding transitions in these multimeric ion channels.
– This technique can be broadly applied to unambiguously quantify binding cooperativity in other membrane receptors.
D. S. White, S. Chowdhury, V. Idikuda, R. Zhang, S. T. Retterer, R. H. Goldsmith, and B. Chanda, "cAMP Binding to Closed Pacemaker Ion Channels is Non-Cooperative," Nature (2021). DOI: 10.1038/s41586-021-03686-x