Abstract
The conformations of biological macromolecules are intimately related to their cellular functions. Conveniently, the well-characterized dipole–dipole distance-dependence of Förster resonance energy transfer (FRET) makes it possible to measure and monitor the nanoscale spatial dimensions of these conformations using fluorescence spectroscopy. For this reason, FRET is often used in conjunction with single-molecule detection to study a wide range of conformationally dynamic biochemical processes. Written for those not yet familiar with the subject, this review aims to introduce biochemists to the methodology associated with single-molecule FRET, with a particular emphasis on how it can be combined with biomolecular simulations to study diverse interactions between nucleic acids and proteins. In the first section, we highlight several conceptual and practical considerations related to this integrative approach. In the second section, we review a few recent research efforts wherein various combinations of single-molecule FRET and biomolecular simulations were used to study the structural and dynamic properties of biochemical systems involving different types of nucleic acids (e.g., DNA and RNA) and proteins (e.g., folded and disordered).
Highlights
Spectroscopic methods are incredibly valuable for biochemists
Forster resonance energy transfer (FRET) is often used in conjunction with single-molecule detection to study a wide range of conformationally dynamic biochemical processes. Written for those not yet familiar with the subject, this review aims to introduce biochemists to the methodology associated with single-molecule FRET, with a particular emphasis on how it can be combined with biomolecular simulations to study diverse interactions between nucleic acids and proteins
We review a few recent research efforts wherein various combinations of single-molecule FRET and biomolecular simulations were used to study the structural and dynamic properties of biochemical systems involving different types of nucleic acids (e.g., DNA and RNA) and proteins
Summary
Spectroscopic methods are incredibly valuable for biochemists. For example, in an absorbance measurement the amount of light transmitted through a sample is used to quantify the optical density, and concentration, of biomolecular chromophores in solution [1]. When important instrumental and experimental correction factors for spectral cross-talk and the nonidentical quantum yields and detection efficiencies of the two fluorophores are known [24,25,26,27,28], FA and FD can be used to more accurately determine the value of E enabling biochemists to quantify the nanoscopic distance, r, between the centers of the two fluorophores with sub-nanometer accuracy [29] In this way, the neighboring donor and acceptor fluorophores of a FRET-labeled biomolecule function together as a spectroscopic ruler [30,31] for attaining nanoscale distances. The spectrally resolved fluorescence from the acceptor (FA) and donor (FD) fluorophores is recorded
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