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Short-range spectroscopic ruler based on a single-molecule optical switch.

ABSTRACT: We demonstrate a novel all-optical switch consisting of two molecules: a primary fluorophore (Cy5) that can be switched between a fluorescent and a dark state by light of different wavelengths, and a secondary chromophore (Cy3) that facilitates switching. The interaction between the two molecules exhibits a distance dependence much steeper than that of conventional Fo rster resonance energy transfer. This enables the switch to act as a ruler with the capability to probe distances difficult to access by other spectroscopic methods, thus presenting a new tool for the study of biomolecules at the single-molecule level.

SUBMITTER: Bates M

PROVIDER: S-EPMC2652517 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Short-range spectroscopic ruler based on a single-molecule optical switch.

Bates Mark M Blosser Timothy R TR Zhuang Xiaowei X

Physical review letters 20050315 10

We demonstrate a novel all-optical switch consisting of two molecules: a primary fluorophore (Cy5) that can be switched between a fluorescent and a dark state by light of different wavelengths, and a secondary chromophore (Cy3) that facilitates switching. The interaction between the two molecules exhibits a distance dependence much steeper than that of conventional Fo rster resonance energy transfer. This enables the switch to act as a ruler with the capability to probe distances difficult to ac ...[more]

PMID: 15783528

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Project description:Förster resonance energy transfer (FRET) is an important mechanism for the estimation of intermolecular distances, e.g., in fluorescent labeled proteins. The interpretations of FRET experiments with standard Förster theory relies on the following approximations: (i) a point-dipole approximation (PDA) for the coupling between transition densities of the chromophores, (ii) a screening of this coupling by the inverse optical dielectric constant of the medium, and (iii) the assumption of fast isotropic sampling over the mutual orientations of the chromophores. These approximations become critical, in particular, at short intermolecular distances, where the PDA and the screening model become invalid and the variation of interchromophore distances, and not just orientations, has a critical influence on the excitation energy transfer. Here, we present a quantum chemical/electrostatic/molecular dynamics (MD) method that goes beyond all of the above approximations. The Poisson-TrEsp method for the ab initio/electrostatic calculation of excitonic couplings in a dielectric medium is combined with all-atom molecular dynamics (MD) simulations to calculate FRET efficiencies. The method is applied to analyze single-molecule experiments on a polyproline helix of variable length labeled with Alexa dyes. Our method provides a quantitative explanation of the overestimation of FRET efficiencies by the standard Förster theory for short interchromophore distances for this system. A detailed analysis of the different levels of approximation that connect the present Poisson-TrEsp/MD method with Förster theory reveals error compensation effects, between the PDA and the neglect of correlations in interchromophore distances and orientations on one hand and the neglect of static disorder in orientations and interchromophore distances on the other. Whereas the first two approximations are found to decrease the FRET efficiency, the latter two overcompensate this decrease and are responsible for the overestimation of the FRET efficiency by Förster theory.

Dataset Information

Short-range spectroscopic ruler based on a single-molecule optical switch.

Publications

Short-range spectroscopic ruler based on a single-molecule optical switch.

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