title: Temperature-cycle microscopy reveals single-molecule conformational heterogeneity.
abstract: Our previous temperature-cycle study reported FRET transitions between different states on FRET-labeled polyprolines [Yuan et al., PCCP, 2011, 13, 1762]. The conformational origin of such transitions, however, was left open. In this work, we apply temperature-cycle microscopy of single FRET-labeled polyproline and dsDNA molecules and compare their responses to resolve the conformational origin of different FRET states. We observe different steady-state FRET distributions and different temperature-cycle responses in the two samples. Our temperature-cycle results on single molecules resemble the results in steady-state measurements but reveal a dark state which could not be observed otherwise. By comparing the timescales and probabilities of different FRET states in temperature-cycle traces, we assign the conformational heterogeneity reflected by different FRET states to linker dynamics, dye-chain and dye-dye interactions. The dark state and low-FRET state are likely due to dye-dye interactions at short separations.
authors: Huygens-KamerlingOnnes Laboratory, Leiden University, Leiden, The Netherlands. orrit@physics.leidenuniv.nl.
authors: Haifeng
authors: H
authors: Yuan
authors: Alexander
authors: A
authors: Gaiduk
authors: Joanna R
authors: JR
authors: Siekierzycka
authors: Satoru
authors: S
authors: Fujiyoshi
authors: Michio
authors: M
authors: Matsushita
authors: Daniel
authors: D
authors: Nettels
authors: Benjamin
authors: B
authors: Schuler
authors: Claus A M
authors: CA
authors: Seidel
authors: Michel
authors: M
authors: Orrit
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D008853:Microscopy / Q000379:methods*; D008968:Molecular Conformation; D013696:Temperature