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

Autor:	Chen Z; Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720., Shaw A; Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720., Wilson H; Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544., Woringer M; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.; Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine Center of Excellence, University of California, Berkeley, CA 94720.; Unité Imagerie et Modélisation, Institut Pasteur, 75015 Paris, France., Darzacq X; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.; Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine Center of Excellence, University of California, Berkeley, CA 94720., Marqusee S; Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; marqusee@berkeley.edu quanw@princeton.edu carlosb@berkeley.edu.; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.; Department of Chemistry, University of California, Berkeley, CA 94720.; Biophysics Graduate Group, University of California, Berkeley, CA 94720., Wang Q; Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; marqusee@berkeley.edu quanw@princeton.edu carlosb@berkeley.edu., Bustamante C; Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; marqusee@berkeley.edu quanw@princeton.edu carlosb@berkeley.edu.; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.; Department of Chemistry, University of California, Berkeley, CA 94720.; Biophysics Graduate Group, University of California, Berkeley, CA 94720.; Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, CA 94720.; Department of Physics, University of California, Berkeley, CA 94720.; Kavli Energy Nanoscience Institute, University of California, Berkeley, CA 94720.; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2020 Sep 01; Vol. 117 (35), pp. 21328-21335. Date of Electronic Publication: 2020 Aug 18.
DOI:	10.1073/pnas.2006900117
Abstrakt:	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 ( p NPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that p NPP-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. Competing Interests: The authors declare no competing interest. (Copyright © 2020 the Author(s). Published by PNAS.)
Databáze:	MEDLINE
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