Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy which can be used to directly monitor the environment and dynamics around a fluorophore in a complex biological system. 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 absorption band, is termed 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 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 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 since hydration plays a crucial modulatory role in a large number of important cellular events, including lipid-protein interactions and ion transport. The interfacial region in membranes, characterized by unique motional and dielectric characteristics, represents an appropriate environment for displaying wavelength-selective fluorescence effects. The application of REES and related techniques (wavelength-selective fluorescence approach) as a powerful tool to monitor the organization and dynamics of probes and peptides bound to membranes, micelles, and reverse micelles is discussed

Monitoring cholesterol organization in membranes at low concentrations utilizing the wavelength-selective fluorescence approach

We previously showed using a fluorescent analogue of cholesterol (NBD-cholesterol, or 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol), that cholesterol may exhibit local organization at low concentrations in membranes by the formation of transbilayer tail-to-tail dimers of cholesterol (Rukmini, R., Rawat, S.S., Biswas, S.C., Chattopadhyay, A., 2001. Biophys. J. 81, 2122-2134). In this report, we have monitored the microenvironmental features of cholesterol monomers and dimers utilizing wavelength-selective fluorescence spectroscopy. Our results utilizing red edge excitation shift (REES) and wavelength-dependent change in fluorescence anisotropy show that the microenvironment around the NBD moieties in the dimer form is more rigid possibly due to steric constraints imposed by the dimer conformation. These results provide new information and are relevant in understanding the organization of cholesterol in membranes at low concentrations

Influence of lipid chain unsaturation on membrane-bound melittin: a fluorescence approach

AbstractMelittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. The organization of membrane-bound melittin is dependent on the physical state and composition of membranes. In particular, polyunsaturated lipids have been shown to modulate the membrane-disruptive action of melittin. Phospholipids with polyunsaturated acyl chains are known to modulate a number of physical properties of membranes and play an important role in regulating structure and function of membrane proteins. In this study, we have used melittin to address the influence of unsaturated lipids in modulating lipid–protein interactions. Our results show that fluorescence parameters such as intensity, emission maximum, polarization, lifetime and acrylamide quenching of melittin incorporated in membranes are dependent on the degree of unsaturation of lipids in membranes. Importantly, melittin in membranes composed of various unsaturated lipids shows red edge excitation shift (REES) implying that melittin is localized in a motionally restricted region in membranes. The extent of REES was found to increase drastically in membranes with increasing unsaturation, especially when the lipids contained more than two double bonds. In addition, increasing unsaturation in membranes causes a considerable change in the secondary structure of membrane-bound melittin. Taken together, our results assume significance in the overall context of the role of unsaturated lipids in membranes in the organization and function of membrane proteins and membrane-active peptides

Melittin: a membrane-active peptide with diverse functions

Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic due to the presence of a stretch of positively charged amino acids. This amphiphilic property of melittin has resulted in melittin being used as a suitable model peptide for monitoring lipid-protein interactions in membranes. In this review, the solution and membrane properties of melittin are highlighted, with an emphasis on melittin-membrane interaction using biophysical approaches. The recent applications of melittin in various cellular processes are discussed

Restricted mobility of the sole tryptophan in membrane-bound melittin

In spite of numerous studies, there appears to be no consensus regarding the orientation and aggregation state of membrane-bound melittin. We report here the restricted environment of the sole tryptophan residue in membrane-bound melittin using environment-induced effects on the rates of solvent relaxation. When incorporated into unilamellar vesicles of dioleoyl-sn-glycero-3-phosphocholine (DOPC), melittin exhibits a red edge excitation shift (REES) of 5 nm. In addition, fluorescence polarization of melittin m the membrane shows both excitation and emission wavelength dependence. Taken together, these observations indicate that the tryptophan residue of melittin is located in a motionally restricted region in the membrane

Influence of cholesterol and ergosterol on membrane dynamics using different fluorescent reporter probes

Ergosterol is an evolutionary precursor of cholesterol and is the major sterol present in lower eukaryotes. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is still at a very early stage. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing fluorescent reporter probes pyrene and TMA-DPH. These results show, for the first time, the important differences on the effect of cholesterol and ergosterol in short-range ordering (reported by TMA-DPH) and long-range dynamics (reported by pyrene). In addition, pyrene vibronic peak intensity ratio provides information on polarity of the microenvironment experienced by the probe. These novel results are relevant in the context of membrane domains in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes

A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin<SUB>1A</SUB> receptor

Insolubility in non-ionic detergents such as Triton X-100 is a widely used biochemical criterion for characterization of membrane domains. We report here a novel green fluorescent protein fluorescence-based approach to directly determine detergent insolubility of specific membrane proteins. We have applied this method to explore the detergent resistance of an important G-protein coupled receptor, the serotonin1A (5-HT1A) receptor. Our results show, for the first time, that a small yet significant fraction of the 5-HT1A receptor exhibits detergent insolubility. These results are validated by control experiments involving fluorescent lipid probes and protein markers. Our results assume relevance in the context of localization of the 5-HT1A receptor in membrane domains and its significance in receptor function and signaling

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

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