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

Abstract

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 ofmore » 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.« less

Authors:
 [1]
  1. Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
813378
Report Number(s):
LBNL-52404
R&D Project: 504901; TRN: US0303901
DOE Contract Number:  
AC03-76SF00098
Resource Type:
Thesis/Dissertation
Resource Relation:
Other Information: TH: Thesis (Ph.D.); Submitted to the Univ. of California, Berkeley, CA (US); PBD: 30 Jul 2002
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSICS; 72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS; ACCURACY; BRIGHTNESS; DATA ACQUISITION; DIFFUSION; FLUORESCENCE; ENERGY TRANSFER; PHOTONS; RESONANCE; SPECTROSCOPY

Citation Formats

Laurence, Ted Alfred. Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions. United States: N. p., 2002. Web. doi:10.2172/813378.
Laurence, Ted Alfred. Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions. United States. doi:10.2172/813378.
Laurence, Ted Alfred. Tue . "Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions". United States. doi:10.2172/813378. https://www.osti.gov/servlets/purl/813378.
@article{osti_813378,
title = {Photon-counting single-molecule spectroscopy for studying conformational dynamics and macromolecular interactions},
author = {Laurence, Ted Alfred},
abstractNote = {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.},
doi = {10.2172/813378},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2002},
month = {1}
}

Thesis/Dissertation:
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