Molecular Dynamics Revealed by Single-Molecule FRET Measurement
Recent advances in single-molecule imaging have resulted in a series of discoveries regarding characteristic behavior and dynamics of individual molecules. Among the single-molecule imaging techniques, fluorescence resonance energy transfer (FRET) measurement is relatively easy to set up, yet is a powerful method; it can visualize substrate binding and dissociation as well as intramolecular structural changes within a single molecule in real time. Here, we first review single-molecule fluorescence imaging techniques that open a way to establish single-molecule FRET (smFRET) measurement. Then, we describe two examples of the characteristic dynamics of individual molecules revealed by smFRET: antibiotic-mediated protein translation inhibition and the intramolecular structural changes in CRISPR-Cas9, a versatile genome-editing tool. Finally, we introduce some of the latest advances in smFRET technique.
KeywordsFluorescence resonance energy transfer Molecular biophysics Single-molecule FRET Aminoglycoside antibiotics Gene-editing techniques CRISPR_Cas9
FRET techniques have been widely used for measuring the dynamics of biomolecules because of its high sensitivity as a nanoscale distance sensor. Between two closely located fluorescent molecules, energy in an excited donor fluorescent probe is resonantly transferred to an adjacent acceptor fluorescent probe, thereby decreasing the donor’s fluorescence intensity and increasing the acceptor’s fluorescence intensity. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between the two fluorescent molecules. Accordingly, FRET is an extremely sensitive measurement system for detecting changes in the distance between two fluorescent probes, particularly around what is called the Förster distance, namely, a distance that yields a FRET efficiency of 0.5 (4–7 nm for a pair of typical fluorescent probes) (Lakowicz 2006). As such, FRET measurement is ideally suited for detecting changes in the distance between domains or subunits within a protein or nucleic acids during conformational changes. Moreover, based on the ratio of fluorescence intensities of two fluorescent molecules, it can achieve high signal-to-noise ratio in measurements of binding and dissociation reactions compared with measurements involving a single fluorescent molecule. These advantages have made FRET an extensively used technique for researching the dynamics of biomolecules.
Conventional bulk FRET measurements, however, only yield mean measurement values of a large number of molecules. Therefore, these measurements are unable to extract information on the distribution of multiple molecules. The development of single-molecule imaging technologies, capable of distinguishing fluorescence intensities from individual molecules, has overcome this limitation. In combination with the single-molecule imaging techniques, FRET measurements are able to distinguish the state of each molecule in real time. This combination has led to the discoveries of diversities in dynamics and states of biomolecules in the same conditions.
In this chapter, we first outlines the single-molecule imaging techniques, which provide the basis for establishing smFRET measurement, then presents examples of protein dynamics research employing smFRET, and finally introduces some state-of-the-art smFRET applications.
10.1 Single-Molecule Fluorescence Imaging
To detect single molecule fluorescent probes using optical microscopy, the techniques to reduce background light combined with an intense illumination and highly sensitive camera systems are required. By the late 1980s, illumination and imaging systems had already been refined to the point of theoretically being able to detect single molecules of fluorescent probes. Nevertheless, the influence of background light from non-observed fluorescent probes had stalled the development of a system capable of identifying fluorescence from a single molecule. In 1990, Shera et al. succeeded in distinguishing fluorescent signals emitted from a single fluorescent probe molecule using pulsed light to induce photoexcitation of a low-concentration fluorescent probe solution streaming through a flow cell (Shera et al. 1990). However, this technique is arguably more pertinent to classifying as flow cytometry rather than microscopic imaging. Later, in 1993, Betzig et al. paved the way for the technology of single-molecule fluorescence imaging by employing near-field scanning optical microscopy (Betzig and Chichester 1993). Their method took advantage of the phenomenon in which illuminating a small hole with a diameter less than the wavelength of the light results in the light emerging only in the immediate vicinity of the hole. By limiting the area of illumination to a minimum, that is, reducing the background light from fluorescent probes outside the observation area, they achieved single-molecule fluorescence imaging. Thereafter, total internal reflection fluorescence microscopy (TIRFM) also has realized single-molecule fluorescence imaging by employing a similar strategy (Funatsu et al. 1995). TIRFM uses evanescent waves that occur in the immediate vicinity (~200 nm) of the interfacial surface between glass and water. Thus, only fluorescent probes present within ~200 nm from the glass surface were illuminated under TIRFM (Axelrod 1981). Compared with the near-field scanning microscopy, TIRFM is easy to set up and eligible for high-speed imaging. TIRFM requires only a standard fluorescence laser microscope with the ability to adjust the incidence angle of excitation light. Moreover, unnecessity of the scanning process makes TIRFM capable to swiftly capture images of an extensive area in a single shot. These two advantages have contributed to the widespread usage of TIRFM in the single-molecule imaging field. Today, modified epifluorescence microscopy (Sase et al. 1995), confocal microscopy (Nie et al. 1994), oblique illumination microscopy (Tokunaga et al. 2008), and light sheet microscopy (Ritter et al. 2010), which have a deeper range of photoexcitation than TIRFM, can also be applied to single-molecule imaging. Furthermore, the availability of increasingly bright commercial light-emitting diodes means that laser lighting equipment is no longer a prerequisite. These technical advances provide many options for building up a single-molecule microscopy appropriate for each experiment.
10.2 Molecular Dynamics of Proteins Measured by smFRET
10.3 Advances in smFRET Methods
The smFRET techniques presented in this chapter have been advancing further by incorporating other methodologies. For instance, attempts are being made to apply luminescent probes to observe single-molecule dynamics. Luminescent probes do not require illumination light, surmounting the obstacles of fluorescence imaging: autofluorescence, photo-damage to the samples and the strict limitations in the use of optogenetic tools. However, low brightness of the luminescent probes has limited the application of luminescent imaging. Recently, several bright and multicolor luminescent probes were developed by employing resonance energy transfer from luminescent probes to fluorescent proteins (Suzuki and Nagai 2017). The developed probes have greatly improved the sensitivity of luminescent imaging. In addition to the luminescent probes, various biosensors using FRET technique have been developed, enabling elucidation of the localization and dynamics of diverse intracellular molecules in single-molecule sensitivities.
Now, efforts are also underway to accelerate the smFRET measurement process. While conventional smFRET is able to detect the dynamics of biomolecules within 10–100 millisecond order, a recent study demonstrated that smFRET can track protein conformational changes at the microsecond level (Otosu et al. 2015). When FRET occurs, the donor fluorescent probe shows a decreased fluorescence intensity as well as a shortened cycle of time before returning to the ground state after photoexcitation (fluorescence lifetime). Compared with conventional intensity measurements, the lifetime measurements require fewer fluorescent photons to monitor FRET efficiency, and hence, can improve the temporal resolution of FRET measurements. The study using this method revealed the detailed process of conformational changes in cytochrome c protein at a sub-microsecond temporal resolution (Otosu et al. 2015).
Although the FRET techniques presented above are premised on the labeling of samples with two (or more) probes in different colors, smFRET can be measured using a fluorescent probe. Using non-fluorescent quenchers allows experimenters to perform an analysis similar to two-colored FRET measurement simply by measuring the fluorescence intensity of one fluorescent probe. When the donor fluorescent probe approaches a quencher, the fluorescence intensity decreases due to resonance energy transfer from the donor to the quencher. Therefore, one can observe conformational changes, bindings, and dissociations of labeled biomolecules simply by measuring the change in fluorescence intensity of the donor probe (Chen et al. 2012). Moreover, not only between heterologous molecules, FRET between homologous probes (referred to as homo-FRET) can be measurable. When illuminated by polarized light, only fluorescent probes in specific orientation are photoexcited and emit polarized fluorescence. If two identical fluorescent probes locate close together, because of FRET between the probes, the probes not in the photo-excitable orientation also emit fluorescence light polarized differently than that from the photoexcited donor probe, resulting in decreased anisotropy of the fluorescence. Therefore, distance changes between identical probes can be monitored by measuring the polarization components of fluorescence. This homo-FRET technique expands the application of FRET measurement in the studies of dynamics of biomolecules which can be labeled with only one-type of fluorescent probe, such as oligomerization processes of an endogenous protein (Bader et al. 2011). These advanced optical techniques have further sophisticated the smFRET method, so that we can elucidate more detailed behaviors of wider range of biomolecules.
smFRET is a particularly useful technique for investigating the behavior of individual biomolecules. Its applications have been steadily expanding by incorporating various other optical technologies. The strength of smFRET lies in its ability to track dynamic conformational changes and bindings/dissociations of ligands under near physiological conditions in real-time. Although smFRET measurements only detect distances and angles between a few positions labeled with FRET probes, this drawback can be complemented by X-ray crystal structure and electron microscopic analyses that visualize entire structures at high resolution. The appropriate combinations of technologies will further deepen our understanding of the molecular basis of biological phenomena.
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