title: Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.
abstract: Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.
authors.affiliation: Institute of Molecular Biology and Department of Chemistry and Biochemistry and ‡Oregon Center for Optical, Molecular and Quantum Science and Department of Chemistry and Biochemistry, University of Oregon , Eugene, Oregon 97403, United States.
authors.forename: Carey
authors.initials: C
authors.lastname: Phelps
authors.affiliation: Institute of Molecular Biology and Department of Chemistry and Biochemistry and ‡Oregon Center for Optical, Molecular and Quantum Science and Department of Chemistry and Biochemistry, University of Oregon , Eugene, Oregon 97403, United States.
authors.forename: Brett
authors.initials: B
authors.lastname: Israels
authors.affiliation: Institute of Molecular Biology and Department of Chemistry and Biochemistry and ‡Oregon Center for Optical, Molecular and Quantum Science and Department of Chemistry and Biochemistry, University of Oregon , Eugene, Oregon 97403, United States.
authors.forename: Morgan C
authors.initials: MC
authors.lastname: Marsh
authors.affiliation: Institute of Molecular Biology and Department of Chemistry and Biochemistry and ‡Oregon Center for Optical, Molecular and Quantum Science and Department of Chemistry and Biochemistry, University of Oregon , Eugene, Oregon 97403, United States.
authors.forename: Peter H
authors.initials: PH
authors.lastname: von Hippel
authors.affiliation: Institute of Molecular Biology and Department of Chemistry and Biochemistry and ‡Oregon Center for Optical, Molecular and Quantum Science and Department of Chemistry and Biochemistry, University of Oregon , Eugene, Oregon 97403, United States.
authors.forename: Andrew H
authors.identifier: 0000-0003-2109-327X
authors.initials: AH
authors.lastname: Marcus
mesh_terms: D017122:Bacteriophage T4 / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D061848:Optical Imaging / Q000379:methods*; D000072760:Single Molecule Imaging / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D014764:Viral Proteins