title: Intramolecular distances and dynamics from the combined photon statistics of single-molecule FRET and photoinduced electron transfer.
abstract: Single-molecule Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) have developed into versatile and complementary methods for probing distances and dynamics in biomolecules. Here we show that the two methods can be combined in one molecule to obtain both accurate distance information and the kinetics of intramolecular contact formation. In a first step, we show that the fluorescent dyes Alexa 488 and Alexa 594, which are frequently used as a donor and acceptor for single-molecule FRET, are also suitable as PET probes with tryptophan as a fluorescence quencher. We then performed combined FRET/PET experiments with FRET donor- and acceptor-labeled polyproline peptides. The placement of a tryptophan residue into the polyglycylserine tail incorporated in the peptides allowed us to measure both FRET efficiencies and the nanosecond dynamics of contact formation between one of the fluorescent dyes and the quencher. Variation of the linker length between the polyproline and the Alexa dyes and in the position of the tryptophan residue demonstrates the sensitivity of this approach. Modeling of the combined photon statistics underlying the combined FRET and PET process enables the accurate analysis of both the resulting transfer efficiency histograms and the nanosecond fluorescence correlation functions. This approach opens up new possibilities for investigating single biomolecules with high spatial and temporal resolution.
authors.affiliation: Department of Biochemistry, University of Zurich , Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Dominik
authors.initials: D
authors.lastname: Haenni
authors.forename: Franziska
authors.initials: F
authors.lastname: Zosel
authors.forename: Luc
authors.initials: L
authors.lastname: Reymond
authors.forename: Daniel
authors.initials: D
authors.lastname: Nettels
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schuler
mesh_terms: D004579:Electron Transport; D004583:Electrons; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017785:Photons; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
chemical_list: C417561:Alexa 488 hydrazide; C417664:Alexa594; D005456:Fluorescent Dyes; D006834:Hydrazines; D009930:Organic Chemicals; D010455:Peptides; C011083:polyproline; D014364:Tryptophan