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

View ORCID ProfileZhijie Chen, View ORCID ProfileAlan Shaw, View ORCID ProfileHugh Wilson, View ORCID ProfileMaxime Woringer, View ORCID ProfileXavier Darzacq, View ORCID ProfileSusan Marqusee, View ORCID ProfileQuan Wang, View ORCID ProfileCarlos Bustamante
doi: https://doi.org/10.1101/2020.04.10.036442
Zhijie Chen
aInstitute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720;
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Alan Shaw
aInstitute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720;
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Hugh Wilson
bLewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544
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Maxime Woringer
cDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
dLi Ka Shing Center for Biomedical and Health Sciences, CIRM Center of Excellence, University of California, Berkeley, Berkeley, CA 94720
eUnité Imagerie et Modélisation, Institut Pasteur, Paris, France
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Xavier Darzacq
cDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
dLi Ka Shing Center for Biomedical and Health Sciences, CIRM Center of Excellence, University of California, Berkeley, Berkeley, CA 94720
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Susan Marqusee
aInstitute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720;
cDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
fDepartment of Chemistry, University of California, Berkeley, CA 94720
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  • For correspondence: marqusee@berkeley.edu quanw@princeton.edu carlosb@berkeley.edu
Quan Wang
bLewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544
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  • For correspondence: marqusee@berkeley.edu quanw@princeton.edu carlosb@berkeley.edu
Carlos Bustamante
aInstitute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720;
cDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
fDepartment of Chemistry, University of California, Berkeley, CA 94720
gJason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, CA
hBiophysics Graduate Group, University of California, Berkeley, Berkeley, CA, USA
iDepartment of Physics, University of California, Berkeley, Berkeley, CA, USA
jKavli Energy Nanoscience Institute, University of California, Berkeley, Berkeley, CA, USA
kHoward Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA
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  • For correspondence: marqusee@berkeley.edu quanw@princeton.edu carlosb@berkeley.edu
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ABSTRACT

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. Experimental observations of catalysis-enhanced enzyme diffusion, to date, 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 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 (SPT), 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 SPT and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

SIGNIFICANCE STATEMENT Recent experiments have suggested that the energy released by a chemical reaction can propel its enzyme catalyst (for example, alkaline phosphatase, ALP). However, this topic remains controversial, partially due to the indirect and ensemble nature of existing measurements. Here, we used recently developed single-molecule approaches to monitor directly the motions of individual proteins in aqueous solution and find that single ALP enzymes do not diffuse faster under catalysis. Instead, we demonstrate that interactions between the fluorescent dye and the enzyme’s substrate can produce the signature of apparent diffusion enhancement in fluorescence correlation spectroscopy (FCS), the standard ensemble assay currently used to study enzyme diffusion and indicate that single-molecule approaches provide a more robust means to investigate diffusion at the nanoscale.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 11, 2020.
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Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments
Zhijie Chen, Alan Shaw, Hugh Wilson, Maxime Woringer, Xavier Darzacq, Susan Marqusee, Quan Wang, Carlos Bustamante
bioRxiv 2020.04.10.036442; doi: https://doi.org/10.1101/2020.04.10.036442
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Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments
Zhijie Chen, Alan Shaw, Hugh Wilson, Maxime Woringer, Xavier Darzacq, Susan Marqusee, Quan Wang, Carlos Bustamante
bioRxiv 2020.04.10.036442; doi: https://doi.org/10.1101/2020.04.10.036442

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