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Single-Molecule Instrumentation and Analyses to Investigate the Calcium Binding Protein Calmodulin
DeVore, Matthew Scott
DeVore, Matthew Scott
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Abstract
Single-molecule methods are being used to understand the structure and dynamics of biomolecules. By avoiding ensemble averaging, these methods investigate the behavior of population subsets allowing for direct observation of conformational distributions and dynamics. In particular, Förster resonance energy transfer (FRET) has been used in conjunction with single-molecule burst measurements to investigate the conformations of biomolecules. In this work, instrumentation and analyses were developed to investigate the FRET states of the protein calmodulin with different dye pairs. First, an alternating laser excitation system was built. Detailed explanations of the instrumental design, hardware, and software which were created to implement this technique are presented. Bayesian approaches for single-molecule burst data were developed. The photon distribution analysis is combined with the classic maximum entropy method and a Monte Carlo sampling approach, called MultiNest, for evidence calculation. The classic maximum entropy method used information entropy in a Bayesian prior to perform a non-parametric, or "model-free", fit to the joint distribution of apparent FRET efficiency and fluorescence photons. The MultiNest approach approximates the fluorescence distribution of single-molecules with the signal distribution and models the apparent FRET efficiency distribution as a sum of Gaussians. For simulated data under ideal conditions, the MultiNest approach with a Gaussian model provided a better model than the classic maximum entropy approach as judged by the Bayesian evidence. However, on experimental single-molecule FRET burst data of dye labeled calmodulin, the classic maximum entropy model was found to be a better than the Gaussian MultiNest model in most cases. The analysis and instruments developed were used to investigate the FRET states displayed by dye labeled calmodulin. It was found that the FRET states were dependent on FRET dye pair. Time-resolved fluorescence lifetime and anisotropy measurements were combined with single-molecule anisotropy measurements to investigate the FRET states and rotational freedom of the dyes. Results from this work indicate that dye-protein interactions may contribute to the FRET states and dynamics measured.
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2012-01-01
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University of Kansas
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DeVore_ku_0099D_12479_DATA_1.pdf
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Keywords
Physical chemistry, Biochemistry, Biophysics, Bayesian Analysis, Calmodulin, FRET, Maximum Entropy, Microscopy, Single-Molecule