Wavelength-selective fluorescence as a novel tool to study organization and dynamics in complex biological systems

Abstract

The dynamics exhibited by a given component of a large macromolecule such as a folded globular protein or an organized supramolecular assembly like the biological membrane is a function of its precise localization within the larger system. A set of approaches based on the red edge effect in fluorescence spectroscopy, which can be used to monitordirectly the environment and dynamics around a fluorophore in a complex biological system, is reviewed in this article. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. This phenomenon arises from the slow rates of solvent relaxation around an excited-state fluorophore, which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise 'optically silent' water molecules. This makes REES and related techniques extremely useful in biology since hydration plays a crucial modulatory role in a large number of important cellular events.

Micellar Organization and Dynamics: A Wavelength-Selective Fluorescence Approach

“…Similar observations have previously been reported for fluorophores in environments of restricted mobility. 15,17,20 Such increasing lifetimes across the emission spectrum may be interpreted in terms of solvent reorientation around the excited-state fluorophore as follows. Observation at shorter wavelengths of emission spectra selects for predominantly unrelaxed fluorophores.…”

“…Such a marked shortening of mean fluorescence lifetime at the red edge of the absorption band is indicative of slow solvent reorientation around the excited state fluorophore. 15,17,20 Table 2 shows the lifetimes of micelle-bound NBD-PE as a function of emission wavelength, keeping the excitation wavelength constant at 465 nm. All decays corresponding to different emission wavelengths could be fitted to biexponential functions.…”

“…The origin of the red edge effect lies in differential extents of solvent reorientation around the excited-state fluorophore, with each excitation wavelength selectively exciting a different average population of fluorophores. 20,21 Since fluorescence lifetime serves as a sensitive indicator for the local environment in which a given fluorophore is placed and is known to be sensitive to excited-state interactions, differential extents of solvent relaxation around a given fluorophore could be expected to give rise to differences in its lifetime. Table 1 shows the lifetimes of NBD-PE in micelles of SDS, Triton X-100, CHAPS, and CTAB as a function of excitation wavelength, keeping the emission wavelength constant.…”

“…[14][15][16][17][18][19] Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy, which can be used to monitor directly the environment and dynamics around a fluorophore in a complex biological system. 20 A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases (refs 20 and 21 and references therein).…”

α-Helix Formation in Melittin and β-Lactoglobulin A Induced by Fluorinated Dialcohols

“…The wavelength of maximum fluorescence emission for a polar fluorophore in a viscous solution or condensed phases with motionally restricted environments has been observed to shift to the red as the excitation wavelength is shifted toward the red edge of the absorption band. [48][49][50][51][52][53][54][55] This REES phenomenon occurs when the dipolar relaxation time for the solvent shell surrounding the fluorophore becomes comparable to or longer than the fluorescence lifetime. As an example, it is known that when melittin is bound to bilayer membranes, it is localized in the motionally restricted interfacial region of the membranes.…”