title: Single-Molecule Analysis of CytochromecFolding by Monitoring the Lifetime of an Attached Fluorescent Probe.
abstract: Conformational dynamics of proteins are important for function. However, obtaining information about specific conformations is difficult for samples displaying heterogeneity. Here, time-resolved fluorescence resonance energy transfer is used to characterize the folding of single cytochromecmolecules. In particular, measurements of the fluorescence lifetimes of individual cytochromecmolecules labeled with a single dye that is quenched by energy transfer to the heme were used to monitor conformational transitions of the protein under partially denaturing conditions. These studies indicate significantly more conformational heterogeneity than has been described previously. Importantly, the use of a purified singly-labeled sample made a direct comparison to ensemble data possible. The distribution of lifetimes of single-proteins was compared to the distribution of lifetimes determined from analysis of ensemble lifetime fluorescence data. The results show broad agreement between single-molecule and ensemble data, with a similar range of lifetimes. However, the single-molecule data reveal greater conformational heterogeneity.
authors.affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, United States.
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Lee
authors.affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, United States.
authors.forename: Wesley B
authors.initials: WB
authors.lastname: Asher
authors.affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, United States.
authors.forename: Harry A
authors.initials: HA
authors.lastname: Stern
authors.affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, United States.
authors.forename: Kara L
authors.initials: KL
authors.lastname: Bren
authors.affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, United States.
authors.forename: Todd D
authors.initials: TD
authors.lastname: Krauss
keywords: change-point algorithm; confocal microscopy; protein folding; single-molecule fluorescence lifetime