Abstract
When the fluorescence intensity of a chromophore attached to or bound in an enzyme relates to a specific reactive step in the enzymatic reaction, a single molecule fluorescence study of the process reveals a time sequence in the fluorescence emission that can be analyzed to derive kinetic and mechanistic information. Reports of various experimental results and corresponding theoretical studies have provided a basis for interpreting these data and understanding the methodology. We have found it useful to parallel experiments with Monte Carlo simulations of potential models hypothesized to describe the reaction kinetics. The simulations can be adapted to include experimental limitations, such as limited data sets, and complexities such as dynamic disorder, where reaction rates appear to change over time. By using models that are known a priori, the simulations reveal some of the challenges of interpreting finite single-molecule data sets by employing various statistical signatures that have been identified.
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Acknowledgements
This work is supported by the National Institutes on Aging (5 R01 AG018017-04), and Michigan Life Science Corridor (MLSC, proposal#1537) to A.G. and D.S. The authors also would like to acknowledge helpful discussion with X. Sunney Xie. The simulation source code written in Mathematica is available for download in our group website at http://www.umich.edu/∼protein/monte.html.
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Shi, J., Gafni, A. & Steel, D. Simulated data sets for single molecule kinetics: some limitations and complications of data analysis. Eur Biophys J 35, 633–645 (2006). https://doi.org/10.1007/s00249-006-0067-5
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DOI: https://doi.org/10.1007/s00249-006-0067-5