Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions

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Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors ... continued below

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196 pages

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Laurence, Ted Alfred July 30, 2002.

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Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors to detect bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract fluorescence resonance energy transfer (FRET) efficiency. Advances in data acquisition and analysis techniques that are providing a more complete picture of the accessible molecular information are discussed. Photon Arrival-time Interval Distribution (PAID) analysis is a new method for monitoring macromolecular interactions by fluorescence detection with simultaneous determination of coincidence, brightness, diffusion time, and occupancy (proportional to concentration) of fluorescently-labeled molecules undergoing diffusion in a confocal detection volume. This method is based on recording the time of arrival of all detected photons, and then plotting the two-dimensional histogram of photon pairs, where one axis is the time interval between each pair of photons 1 and 2, and the second axis is the number of other photons detected in the time interval between photons 1 and 2. PAID is related to Fluorescence Correlation Spectroscopy (FCS) by a collapse of this histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS by measuring the brightness of fluorescent species. A data-fitting model is developed, which is used to simultaneously determine coincidence, brightness, diffusion time, and occupancy from experiments performed on fluorophore-labeled dsDNA test samples. Using simulations, the performance of PAID is compared with existing methods. The statistical accuracy of the parameters extracted using PAID exceeds or matches the accuracy of the other methods, while providing additional information.

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196 pages

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INIS; OSTI as DE00813378

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  • Other Information: TH: Thesis (Ph.D.); Submitted to the Univ. of California, Berkeley, CA (US)

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  • Report No.: LBNL--52404
  • Grant Number: AC03-76SF00098
  • Office of Scientific & Technical Information Report Number: 813378
  • Archival Resource Key: ark:/67531/metadc740578

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  • July 30, 2002

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  • Oct. 18, 2015, 6:40 p.m.

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  • April 4, 2016, 1:55 p.m.

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Laurence, Ted Alfred. Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions, thesis or dissertation, July 30, 2002; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc740578/: accessed September 21, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.