Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

Abstract

A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a corresponding 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 viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which depends on 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 extremely useful since hydration plays a crucial modulatory role in the formation and maintenance of organized molecular assemblies such as folded proteins in aqueous solutions and biological membranes. The application of REES as a powerful tool to monitor the organization and dynamics of a variety of soluble, cytoskeletal, and membrane-bound proteins is discussed.

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

“…It has been shown that a change of a few nanometers in REES is significant, and the observed changes in the magnitude of REES (0-6 nm) in our case fall within the range of expected REES changes (∼5 nm) associated with changes from fully restricted to freely relaxing environments (24,25). It should be mentioned that REES does not generally provide a quantitative estimate of the water relaxation dynamics because the magnitude of REES is not always linear and is dependent on the given system under investigation (22). In WT KcsA, the solvent relaxation rate is considerably slower around most of the residues during inactivation gating (residues Arg52, Gly53, Ala54, Gln58, and Ile60 in the outer loop and Leu81, Tyr82, and Val84 in the inner loop).…”

Control of human VDAC-2 scaffold dynamics by interfacial tryptophans is position specific

“…These residues help in docking the transmembrane segment of the protein within the membrane due to their ability of ‘snorkeling’ at the interface [1], [2]. Tryptophans and tyrosines often form an ‘aromatic belt’ in membrane proteins that stabilize the native structure of the protein, and contribute immensely to the overall protein stability [2], [3], [4]. Considering the high cost of tryptophan synthesis in the cell, particularly in lower organisms [5], the indole moiety is incorporated judiciously in proteins.…”

Organization and Dynamics of Tryptophan Residues in Brain Spectrin: Novel Insight into Conformational Flexibility

“…The dipolar relaxation time of the solvent shell around a polar fluorophore becomes comparable to or longer than its fluorescence lifetime in motionally restricted environment and this gives rise to photoselection of differentially relaxed population of fluorophores upon excitation toward the red edge [39][40][41][42][43][44]. The fact that the fluorophore in REES measurements merely acts as a reporter group and allows to monitor the mobility parameters of the environment itself (represented by the relaxing solvent molecules) adds to its uniqueness.…”