Simulated data sets for single molecule kinetics: some limitations and complications of data analysis |
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Authors: | Jue Shi Ari Gafni Duncan Steel |
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Institution: | (1) Biophysics Research Division, University of Michigan, Ann Arbor, MI 48109, USA;(2) Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA;(3) Department of Physics and EECS, University of Michigan, Ann Arbor, MI 48109, USA;(4) Present address: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA |
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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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