Fluorescence resonance energy transfer FRET experiments

Fluorescence resonance energy transfer (FRET) experiments commonly use the fluorescent spectrum and relaxation times of the Forster donor and acceptor chromophores to find the distances between fluorescent dyes at labeled sites in protein, DNA, RNA, etc. FRET is a type of spectroscopic ruler . The computation uses either experimental quantum yields or relaxation lifetimes to calculate the efficiency of resonance energy transfer Ej. 

Chirio-Lebrun MC, Prats M. Fluorescence resonance energy transfer (FRET) theory and experiments. Biochemical Education 1998, 26, 320-323. 

The elucidation of the structure, dynamics and self assembly of biopolymers has been the subject of many experimental, theoretical and computational studies over the last several decades. [1, 2] More recently, powerful singlemolecule (SM) techniques have emerged which make it possible to explore those questions with an unprecedented level of detail. [3-55] SM fluorescence resonance energy transfer (FRET), [56-60] in particular, has been established as a unique probe of conformational structure and dynamics. [26-55] In those SM-FRET experiments, one measures the efficiency of energy transfer between a donor dye molecule and an acceptor dye molecule, which label specific sites of a macromolecule. The rate constant for FRET from donor to acceptor is assumed to be given by the Forster theory, namely [59,61-64]... 

Forster s theory [1], has enabled the efficiency of EET to be predicted and analyzed. The significance of Forster s formulation is evinced by the numerous and diverse areas of study that have been impacted by his paper. This predictive theory was turned on its head by Stryer and Haugland [17], who showed that distances in the range of 2-50 nm between molecular tags in a protein could be measured by a spectroscopic ruler known as fluorescence resonance energy transfer (FRET). Similar kinds of experiments have been employed to analyze the structure and dynamics of interfaces in blends of polymers. 

The first set of experiments involved fluorescence resonance energy transfer (FRET) between the naphthalene and pyrene-laheled polymers. A 5 1 mixture of PNIPAM-Py to PNIPAM-Na was used. When assembled in micelles, the pyrene acts as a quencher to the naphthalene, leading to high pyrene fluorescence and low naphthalene fluorescence. When the mixture is added to DMPC (liquid phase) or DSPC (gel phase) vesicles at room temperature, naphthalene fluorescence is increased, while pyrene fluorescence is dramatically decreased. This effect is not seen with the PNIPAM-Py-Na polymer, so the reduction in FRET is not due to the hydrophobic environment. This means that the hydrophobic anchors of the PNIPAM-Py and the PNIPAM-Na likely enter the membrane and the dyes are moved apart from one another. The fact that the anchor appeared to insert into the gel-phase DSPC membrane was somewhat surprising. The authors attribute the effect to defects between crystalline domains in the membrane. To test if the LCST transition still occurs when the polymers are anchored to the membrane, differential scanning calorimetry (DFC) was used. The LCST transition of the PNIPAM-Py/PNIPAM-Na mixture in solution was observed in the DFC ttace. When combined with DSPC or DMPC vesicles, the same peak was observed, indicating that the transition does indeed stiU occur, even in the presence of the lipid. 

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