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Single molecule fluorescence resonance energy transfer (or smFRET) is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected on a single molecule rather than on different molecules as in ensemble FRET.

Methodology

Single molecule FRET measurements are typically performed on fluorescence microscopes, either using surface-immobilized or freely-diffusing molecules. Single FRET pairs are illuminated using intense light sources, typically lasers, in order to generate sufficient fluorescence signal to enable single molecule detection. Wide-field multiphoton microscopy is typically combined with total internal reflection fluorescence microscope (TIRF). This selectively excites FRET pairs on the surface of the measurement chamber and rejects noise from the bulk of the sample. Conversely, confocal microscopy minimizes background by focusing the fluorescence light onto a pinhole to reject out of focus light. The confocal volume has a diameter of around 220 nm, and therefore it must be scanned across the sample in order to generate an image. With confocal excitation, it is possible to measure much deeper into the sample than when using TIRF. Fluorescence signal is detected either using ultra sensitive CCD or scientific CMOS cameras for wide field microscopy or SPADs for confocal microscopy. Once the single molecule intensities vs. time are available the FRET efficiency can be computed for each FRET pair as a function of time and thereby it is possible to follow kinetic events on the single molecule scale and to build FRET histograms showing the distribution of states in each molecule. However, data from many FRET pairs must be recorded and combined in order to obtain general information about a sample.

Surface-Immobilized

In surface-immobilized experiments, biomolecules labeled with fluorescent tags are bound to the surface of the coverglass and images of fluorescence are acquired (typically by a CCD or scientific CMOS cameras). Data collection with cameras will produce movies of the specimen which must be processed to derive the single molecule intensities with time.

Freely-Diffusing

SmFRET can also be used to study the conformations of freely diffusing macromolecules on surfaces. In freely-diffusing experiments, the same biomolecules are free to diffuse in solution while being excited by a small excitation volume (usually a diffraction-limited spot). Bursts of photons due a single-molecule crossing the excitation spot are acquired with SPAD detectors. The confocal spot is kept at a fixed position (no scanning happens) and no image is acquired. Instead, the fluorescence photons emitted by individual molecules crossing the excitation volume are recorded and accumulated in order to build a distribution of different populations present in the sample. Measurements employing SPADs can acquire photon timestamps and therefore more directly yield time traces of intensity vs. time.

Advantages of smFRET

SmFRET allows for a more precise analysis of heterogeneous populations and has a few advantages when compared to ensemble FRET.

Ensemble FRET analysis of a heterogeneous population

One benefit of studying distances in single molecules is that heterogeneous populations can be studied more accurately with values specific for each molecule rather than computing an average based on an ensemble. This allows for the study of specific homogeneous populations within a heterogeneous population. For example, if two existing homologous populations within a heterogeneous population have different FRET values, an ensemble FRET analysis will produce a weighted averaged FRET value to represent the population as a whole. Thus, the obtained FRET value does not produce data on the two distinct populations. In contrast, smFRET would be able to differentiate between the two populations and would allow analysis of the existing homologous populations.

SmFRET also provides dynamic temporal resolution of an individual molecule that cannot be accomplished through ensemble FRET measurements. This allows smFRET to be used to study an RNA’s folding dynamics. Similar to protein folding, RNA folding goes through multiple interactions, folding pathways, and intermediates before reaching its native state. Ensemble FRET has the ability to detect well-populated transition states that accumulate in a population, but it lacks the ability to characterize intermediates that are short-lived and do not accumulate. This limit is addressed by smFRET which offers a direct way to observe the intermediates of single molecules regardless of accumulation. Therefore, smFRET demonstrates the ability to capture transient subpopulations in a heterogenous environment.

SmFRET is also shown to utilize three-color system better than ensemble FRET. Three-color system is an extended ability of the original two-color system. Using two acceptor fluorophores rather than one, FRET technique can simultaneously measure two changes in distance. This offers the added advantages of observing for correlated movements and spatial changes of any complex molecule. Ensemble FRET can use three-color system as well, but any obvious advantages are outweighed by three-color system’s requirements, which include a clear separation of fluorophore signals. For a clear distinction of signal, FRET overlaps must be small which also weaken FRET strength. SmFRET corrects this limitation by only targeting single molecules.

Applications

A major application of smFRET is to analyze the minute biochemical nuances that facilitate protein folding. In recent years, multiple techniques have been developed to investigate single molecule interactions that are involved in protein folding and unfolding. Force-probe techniques, using atomic force microscopy and laser tweezers, have provided information on protein stability. smFRET allows researchers to investigate molecular interactions using fluorescence. Forster resonance energy transfer (FRET) was first applied to single molecules by Ha et al. and applied to protein folding in work by Hochstrasser, Weiss, et al. The benefit that smFRET as a whole has afforded to analyzing molecular interactions is the ability to test single molecule interactions directly without having to average ensembles of data. In protein folding analysis, ensemble experiments involve taking measurements of multiple proteins that are in various states of transition between their folded and unfolded state. When averaged, the protein structure that can be inferred from the ensemble of data only provides a rudimentary structural model of protein folding. However, true understanding of protein folding requires deciphering the sequence of structural events along the folding pathways between the folded and unfolded states. It is this particular branch of research that smFRET is highly applicable.

FRET studies calculate corresponding FRET efficiencies as a result of time-resolved observation of protein folding events. These FRET efficiencies can then be used to infer distances between molecules as a function against time. As the protein transitions between the folded and unfolded states, the corresponding distances between molecules can indicate the sequence of molecular interactions that lead to protein folding.

Single-molecule FRET can also be applied to study the conformational changes of the relevant channel motifs in certain channels. For example, labeled tetrameric KirBac potassium channels were labeled with donor and acceptor fluorophores at particular sites in order to understand the structural dynamics within the lipid membrane, thus allowing them to generalize similar dynamics for similar motifs in other eukaryotic Kir channels or even cation channels in general. The use of smFRET in this experiment allowed them to visualize the conformational changes that cannot be seen if the macroscopic measurements were simply averaged, which would lead to looking at an ensemble rather than individual molecules and the conformational changes within, ultimately allowing us to generalize similar dynamics for similar motifs in other eukaryotic channels.

The structural dynamics of the KirBac channel was thoroughly analyzed in both the open and closed states, dependent on the presence of the ligand PIP2. Part of the results based on smFRET demonstrated the structural rigidity of the extracellular region. The selectivity filter and the outer loop of the selectivity filter region was labeled with fluorophores and conformational coupling was observed. The individual smFRET trajectories strongly demonstrated a FRET efficiency of around 0.8 with no fluctuations, regardless of the state of the channel.

Limitations

Despite making approximate estimates, a limitation of smFRET is the difficulty of obtaining the correct distance involved in energy transfer. Requiring an accurate distance estimate gives rise to a major challenge because the fluorescence of the donor and acceptor fluorophores as well as the energy transfer is dependent on the environment and how the dyes are oriented, which can vary depending on the flexibility of where the fluorophores are bound. This issue, however, is not particularly relevant when the distance estimation of the two fluorophores does not need to be determined with exact and absolute precision.

External links

• Schwartz Research Group, CU-Boulder

Category:Scientific techniques