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Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon...

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Autores principales: Chen, Zhijie, Shaw, Alan, Wilson, Hugh, Woringer, Maxime, Darzacq, Xavier, Marqusee, Susan, Wang, Quan, Bustamante, Carlos
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474647/
https://www.ncbi.nlm.nih.gov/pubmed/32817484
http://dx.doi.org/10.1073/pnas.2006900117
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author Chen, Zhijie
Shaw, Alan
Wilson, Hugh
Woringer, Maxime
Darzacq, Xavier
Marqusee, Susan
Wang, Quan
Bustamante, Carlos
author_facet Chen, Zhijie
Shaw, Alan
Wilson, Hugh
Woringer, Maxime
Darzacq, Xavier
Marqusee, Susan
Wang, Quan
Bustamante, Carlos
author_sort Chen, Zhijie
collection PubMed
description Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that pNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.
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spelling pubmed-74746472020-09-18 Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments Chen, Zhijie Shaw, Alan Wilson, Hugh Woringer, Maxime Darzacq, Xavier Marqusee, Susan Wang, Quan Bustamante, Carlos Proc Natl Acad Sci U S A Biological Sciences Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that pNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale. National Academy of Sciences 2020-09-01 2020-08-18 /pmc/articles/PMC7474647/ /pubmed/32817484 http://dx.doi.org/10.1073/pnas.2006900117 Text en Copyright © 2020 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/ https://creativecommons.org/licenses/by-nc-nd/4.0/This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Chen, Zhijie
Shaw, Alan
Wilson, Hugh
Woringer, Maxime
Darzacq, Xavier
Marqusee, Susan
Wang, Quan
Bustamante, Carlos
Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments
title Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments
title_full Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments
title_fullStr Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments
title_full_unstemmed Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments
title_short Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments
title_sort single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by fcs experiments
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474647/
https://www.ncbi.nlm.nih.gov/pubmed/32817484
http://dx.doi.org/10.1073/pnas.2006900117
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