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Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel
Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these d...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8578382/ https://www.ncbi.nlm.nih.gov/pubmed/34753946 http://dx.doi.org/10.1038/s41467-021-26816-5 |
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author | Patel, Vishal R. Salinas, Arturo M. Qi, Darong Gupta, Shipra Sidote, David J. Goldschen-Ohm, Marcel P. |
author_facet | Patel, Vishal R. Salinas, Arturo M. Qi, Darong Gupta, Shipra Sidote, David J. Goldschen-Ohm, Marcel P. |
author_sort | Patel, Vishal R. |
collection | PubMed |
description | Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane. |
format | Online Article Text |
id | pubmed-8578382 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-85783822021-11-15 Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel Patel, Vishal R. Salinas, Arturo M. Qi, Darong Gupta, Shipra Sidote, David J. Goldschen-Ohm, Marcel P. Nat Commun Article Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane. Nature Publishing Group UK 2021-11-09 /pmc/articles/PMC8578382/ /pubmed/34753946 http://dx.doi.org/10.1038/s41467-021-26816-5 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Patel, Vishal R. Salinas, Arturo M. Qi, Darong Gupta, Shipra Sidote, David J. Goldschen-Ohm, Marcel P. Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel |
title | Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel |
title_full | Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel |
title_fullStr | Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel |
title_full_unstemmed | Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel |
title_short | Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel |
title_sort | single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8578382/ https://www.ncbi.nlm.nih.gov/pubmed/34753946 http://dx.doi.org/10.1038/s41467-021-26816-5 |
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