Dataset Information

High-Resolution Single-Molecule FRET via DNA eXchange (FRET X).

ABSTRACT: Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the pair distance with subnanometer precision. The distance between other pairs are determined by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET X technology will be a tool for the high-resolution analysis of biomolecules and nanostructures.

SUBMITTER: Filius M

PROVIDER: S-EPMC8050827 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Publications

High-Resolution Single-Molecule FRET via DNA eXchange (FRET X).

Filius Mike M Kim Sung Hyun SH Severins Ivo I Joo Chirlmin C

Nano letters 20210319 7

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET e ...[more]

PMID: 33739111

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Project description:Proteins are dynamic systems whose structural preferences determine their function. Unfortunately, building atomically detailed models of protein structural ensembles remains challenging, limiting our understanding of the relationships between sequence, structure, and function. Combining single molecule Förster resonance energy transfer (smFRET) experiments with molecular dynamics simulations could provide experimentally grounded, all-atom models of a protein's structural ensemble. However, agreement between the two techniques is often insufficient to achieve this goal. Here, we explore whether accounting for important experimental details like averaging across structures sampled during a given smFRET measurement is responsible for this apparent discrepancy. We present an approach to account for this time-averaging by leveraging the kinetic information available from Markov state models of a protein's dynamics. This allows us to accurately assess which time scales are averaged during an experiment. We find this approach significantly improves agreement between simulations and experiments in proteins with varying degrees of dynamics, including the well-ordered protein T4 lysozyme, the partially disordered protein apolipoprotein E (ApoE), and a disordered amyloid protein (Aβ40). We find evidence for hidden states that are not apparent in smFRET experiments because of time averaging with other structures, akin to states in fast exchange in nuclear magnetic resonance, and evaluate different force fields. Finally, we show how remaining discrepancies between computations and experiments can be used to guide additional simulations and build structural models for states that were previously unaccounted for. We expect our approach will enable combining simulations and experiments to understand the link between sequence, structure, and function in many settings. Understanding protein dynamics is crucial for understanding protein function, yet few methodologies report on protein motion at an atomic level. Combining single molecule Förster resonance energy transfer (smFRET) experiments with computer simulations could provide atomistic models of protein ensembles which are grounded in experiments, however, there has been limited agreement between the two methods to date. Here, we present an algorithm to recapitulate smFRET experiments from molecular dynamics simulations. This approach significantly improves agreement between simulations and experiments for proteins across the ordered spectrum. Moreover, with this approach we can confidently create atomic models for states observed during smFRET experiments which were otherwise difficult to model due to high amounts of flexibility, disorder, or large deviations from crystal-like states.

Dataset Information

High-Resolution Single-Molecule FRET via DNA eXchange (FRET X).

Publications

High-Resolution Single-Molecule FRET via DNA eXchange (FRET X).

Similar Datasets

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