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Diffusion Measurements of Swimming Enzymes with Fluorescence Correlation Spectroscopy

2018

Article

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ConspectusSelf-propelled chemical motors are chemically powered micro- or nanosized swimmers. The energy required for these motors’ active motion derives from catalytic chemical reactions and the transformation of a fuel dissolved in the solution. While self-propulsion is now well established for larger particles, it is still unclear if enzymes, nature’s nanometer-sized catalysts, are potentially also self-powered nanomotors. Because of its small size, any increase in an enzyme’s diffusion due to active self-propulsion must be observed on top of the enzyme’s passive Brownian motion, which dominates at this scale. Fluorescence correlation spectroscopy (FCS) is a sensitive method to quantify the diffusion properties of single fluorescently labeled molecules in solution. FCS experiments have shown a general increase in the diffusion constant of a number of enzymes when the enzyme is catalytically active. Diffusion enhancements after addition of the enzyme’s substrate (and sometimes its inhibitor) of up to 80\% have been reported, which is at least 1 order of magnitude higher than what theory would predict. However, many factors contribute to the FCS signal and in particular the shape of the autocorrelation function, which underlies diffusion measurements by fluorescence correlation spectroscopy. These effects need to be considered to establish if and by how much the catalytic activity changes an enzyme’s diffusion.We carefully review phenomena that can play a role in FCS experiments and the determination of enzyme diffusion, including the dissociation of enzyme oligomers upon interaction with the substrate, surface binding of the enzyme to glass during the experiment, conformational changes upon binding, and quenching of the fluorophore. We show that these effects can cause changes in the FCS signal that behave similar to an increase in diffusion. However, in the case of the enzymes F1-ATPase and alkaline phosphatase, we demonstrate that there is no measurable increase in enzyme diffusion. Rather, dissociation and conformational changes account for the changes in the FCS signal in the former and fluorophore quenching in the latter. Within the experimental accuracy of our FCS measurements, we do not observe any change in diffusion due to activity for the enzymes we have investigated.We suggest useful control experiments and additional tests for future FCS experiments that should help establish if the observed diffusion enhancement is real or if it is due to an experimental or data analysis artifact. We show that fluorescence lifetime and mean intensity measurements are essential in order to identify the nature of the observed changes in the autocorrelation function. While it is clear from theory that chemically active enzymes should also act as self-propelled nanomotors, our FCS measurements show that the associated increase in diffusion is much smaller than previously reported. Further experiments are needed to quantify the contribution of the enzymes’ catalytic activity to their self-propulsion. We hope that our findings help to establish a useful protocol for future FCS studies in this field and help establish by how much the diffusion of an enzyme is enhanced through catalytic activity.

Author(s): Günther, Jan-Philipp and Börsch, Michael and Fischer, Peer
Journal: Accounts of Chemical Research
Year: 2018
Month: August
Day: 30

Department(s): Micro, Nano, and Molecular Systems
Bibtex Type: Article (article)

DOI: 10.1021/acs.accounts.8b00276
Eprint: https://doi.org/10.1021/acs.accounts.8b00276
Note: PMID: 30160941
State: Published
URL: https://doi.org/10.1021/acs.accounts.8b00276

BibTex

@article{doi:10.1021/acs.accounts.8b00276,
  title = {Diffusion Measurements of Swimming Enzymes with Fluorescence Correlation Spectroscopy},
  author = {G{\"u}nther, Jan-Philipp and B{\"o}rsch, Michael and Fischer, Peer},
  journal = {Accounts of Chemical Research},
  month = aug,
  year = {2018},
  note = {PMID: 30160941},
  eprint = {  https://doi.org/10.1021/acs.accounts.8b00276 },
  url = {  https://doi.org/10.1021/acs.accounts.8b00276 },
  month_numeric = {8}
}