The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid-protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media.

We have monitored the membrane-bound channel and nonchannel conformations of gramicidin utilizing red-edge excitation shift (REES), and related fluorescence parameters. In particular, we have used fluorescence lifetime, polarization, quenching, chemical modification, and membrane penetration depth analysis in addition to REES measurements to distinguish these two conformations. Our results show that REES of gramicidin tryptophans can be effectively used to distinguish conformations of membrane-bound gramicidin. The interfacially localized tryptophans in the channel conformation display REES of 7 nm whereas the tryptophans in the nonchannel conformation exhibit REES of 2 nm which highlights the difference in their average environments in terms of localization in the membrane. This is supported by tryptophan penetration depth measurements using the parallax method and fluorescence lifetime and polarization measurements. Further differences in the average tryptophan microenvironments in the two conformations are brought out by fluorescence quenching experiments using acrylamide and chemical modification of the tryptophans by N-bromosuccinimide. In summary, we report novel fluorescence-based approaches to monitor conformations of this important ion channel peptide. Our results offer vital information on the organization and dynamics of the functionally important tryptophan residues in gramicidin.

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.

Background: Protein folding in cells is intimately coupled to protein synthesis. Results: GFP and RFP folding involves slow, cell autonomous insertion of the last ␤-strand into a stable cotranslational folding intermediate. Conclusion:Fluorescent ␤-barrel proteins utilize kinetically coupled co-and post-translational folding strategies. Significance: Specialized folding properties of GFP and RFP facilitate fluorescence acquisition in diverse biological environments.

We have investigated the organization and dynamics of the functionally important tryptophan residues of erythroid spectrin in native and denatured conditions utilizing the wavelength-selective fluorescence approach. We observed a red edge excitation shift (REES) of 4 nm for the tryptophans in the case of spectrin in its native state. This indicates that tryptophans in spectrin are localized in a microenvironment of restricted mobility, and that the regions surrounding the spectrin tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited state tryptophans. Interestingly, spectrin exhibits a REES of 3 nm even when denatured in 8 M urea. This represents the first report of a denatured protein displaying REES. Observation of REES in the denatured state implies that some of the structural and dynamic features of this microenvironment around the spectrin tryptophans are retained even when the protein is denatured. Fluorescence quenching data of denatured spectrin support this conclusion. In addition, we have deduced the organization and dynamics of the hydrophobic binding site of the polarity-sensitive fluorescent probe PRODAN that binds erythroid spectrin with high affinity. When bound to spectrin, PRODAN exhibits a REES of 9 nm. Because PRODAN binds to a hydrophobic site in spectrin, such a result would directly imply that this region of spectrin offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. The results of our study could provide vital insight into the role of tryptophans in the stability and folding of spectrin.

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The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been used extensively to study the organization, dynamics, and function of membrane-spanning channels. The tryptophan residues in gramicidin channels are crucial for maintaining the structure and function of the channel. We explored the structural basis for the reduction in channel conductance in the case of single-tryptophan analogs of gramicidin with three Trp --> hydrophobic substitutions using a combination of fluorescence approaches, which include red edge excitation shift and membrane penetration depth analysis, size-exclusion chromatography, and circular dichroism spectroscopy. We show here that the gramicidin analogs containing single-tryptophan residues adopt a mixture of nonchannel and channel conformations, as evident from analysis of membrane penetration depth, size-exclusion chromatography, and backbone circular dichroism data. These results are potentially useful in analyzing the effect of tryptophan substitution on the functioning of other ion channels and membrane proteins.

We report here depth-dependent solvent relaxation effects in the reverse micellar assembly using the deeply embedded probe NBD-cholesterol, a fluorescent cholesterol analogue in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group is covalently attached to the flexible acyl chain of cholesterol. Because of its deeper location, the NBD group of NBD-cholesterol is capable of reporting solvation dynamics in the deeper regions of the organized molecular assembly in which it is incorporated. NBD-cholesterol exhibits red edge excitation shift (REES) when incorporated into reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane with varying [water]/[surfactant] molar ratio (w o ). Interestingly, the extent of REES increases with increasing w o implying that the overall motional restriction experienced by the reorienting solvent molecules is increased with increasing hydration. This is in contrast to the behavior of interfacially localized probes. In addition, our results show that with increasing w o , the NBD group of NBD-cholesterol experiences increased polarity as evidenced by the decrease in fluorescence lifetime and other fluorescence parameters such as fluorescence intensity. NBD-cholesterol could prove to be a useful probe for monitoring depthdependent dynamics in organized molecular assemblies.