Tryptophan Octyl Ester Research Articles

Validation of depth-dependent fluorescence quenching in membranes by molecular dynamics simulation of tryptophan octyl ester in POPC bilayer.

Depth-dependent fluorescence quenching is an important tool for studying the penetration of proteins and peptides into lipid bilayers. Extracting quantitative information from quenching data is, however, complicated by (1) a limited number of experimentally available quenchers and (2) thermal disorder resulting in broad distributions of the transverse positions of both quenchers and fluorophores. Here we validate and refine a general approach to determining the location of a fluorescent probe along the bilayer normal from quenching data, based on a molecular dynamics (MD) simulation of a model compound, tryptophan octyl ester (TOE), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The TOE ring was found to lie deeply within the bilayer (most probable position of 13.3 Å and center-of-weight of the distribution of 14.8 Å from the bilayer center), and it was very broadly distributed (with 9 Å depth distribution width), which is consistent with previous experimental observations. The depth-dependent quenching profiles were simulated by treating carbon atoms of the lipid acyl chain of POPC as "pseudo-quenchers" and calculating appropriate transverse overlaps and collision rates with indole atoms of TOE. These simulated quenching profiles were well fitted by a Gaussian function of depth, as is routinely done with experimental data subjected to the distribution analysis procedure [Methods Enzymol. 1997, 278, 462-473]. Comparison of the collisional pseudoquenching profiles with the actual profiles of the indole moiety of TOE allows for testing of the validity of the data analysis and identification of the possible sources of error in calculating depths of membrane penetration from quenching data.

A Kinetic Study of the Photodynamic Effect on Tryptophan Methyl Ester and Tryptophan Octyl Ester in DOPC Vesicles

The photodynamic effect on tryptophan methyl ester (trpME) and tryptophan octyl ester (trpOE), using the O(2)((1)Delta(g))-photosensitizers Rose Bengal (RB) and Perinaphthenone (PN) has been studied in large unilamellar vesicles (LUVs) of the phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC) by stationary photolysis and time-resolved methods. This work reports on the influence of both the site (O(2)((1)Delta(g))) generation and the location of the tryptophan derivatives (trpD), on the photo-oxidation process in a compartmentalized system. The apparent rate constant values for chemical quenching of O(2)((1)Delta(g)) by trpOE (k(r,app)), was higher in vesicles than in water. Also, the ratio between apparent reactive and overall rate constant values for the deactivation of O(2)((1)Delta(g)) (k(r,app)/k(t,app)), increases in vesicles as compared with water, when the oxidative species is generated in the lipidic region or at the interface. Nevertheless, this quotient is lower than the corresponding value in water when O(2)((1)Delta(g)) is generated in the aqueous pseudophase. For trpME, the k(r,app)/k(t,app)values in vesicles and in water are quite similar, confirming the fact that trpME is located in the water pseudophase. Results are discussed in terms of relative protection against O(2)((1)Delta(g)) attack in a microheterogeneous medium as compared with water.

Hydrogen Bonding and Solvent Polarity Markers in the UV Resonance Raman Spectrum of Tryptophan: Application to Membrane Proteins

Ultraviolet resonance Raman (UVRR) spectra of tryptophan compounds in various solvents and a model peptide are presented and reveal systematic changes that reflect solvent polarity, hydrogen bond strength, and cation-pi interaction. The commonly utilized UVRR spectral marker for environment polarity that has been based on off-resonance Raman data, the tryptophan Fermi doublet ratio I1360/I1340, exhibits different values in on- and off-resonance Raman spectra as well as for different tryptophan derivatives. Specifically, the UVRR Fermi doublet ratio for indole ranges from 0.3 in polar solvents to 0.8 in nonpolar solvents, whereas the respective values reported here and previously for off-resonance Raman spectra are 0.5-1.3. UVRR Fermi doublet ratios for the more biologically relevant molecule, N-acetyl tryptophan ethyl ester (NATEE), are in a smaller range of 1.1 (polar solvent) to 1.7 (nonpolar solvent) and correlate to the solvent polarity/polarization parameters pi* and ETN. As has been reported previously, several UVRR modes are also sensitive to the hydrogen bond strength of the indole N-H moiety. Here, we report a new unambiguous marker for H-bonding: the ratio of the W10 (approximately 1237 cm-1) intensity to that of the W9 (approximately 1254 cm-1) mode (RW10). This ratio is 0.7 for NATEE in the absence of hydrogen bond acceptors and increases to 3.1 in the presence of strong hydrogen bond acceptors, with a value of 2.3 in water. The W8 and W17 modes shift more than +10 and approximately -5 cm-1 upon increase in hydrogen bond strength; this range for W17 is smaller than that reported previously and reflects a more realistic range for proteins and peptides in solution. Finally, our data provide evidence for change in the W18 and W16 relative intensity in the presence of cation-pi interactions. These UVRR markers are utilized to interpret spectra of model membrane-bound systems tryptophan octyl ester and the peptide toxin melittin. These spectra reveal the importance of intra- and intermolecular hydrogen bonding and cation-pi interactions that likely influence the partitioning of membrane-associated biomolecules to lipid bilayers or self-associated soluble oligomers. The UVRR analysis presented here modifies and augments prior reports and provides an unambiguous set of spectral makers that can be applied to elucidate the molecular microenvironment and structure of a wide range of complex systems, including anchoring tryptophan residues in membrane proteins and peptides.

Effect of structural transition of the host assembly on dynamics of a membrane-bound tryptophan analogue

Tryptophan octyl ester (TOE) represents an important model for membrane-bound tryptophan residues. In this article, we have explored the effect of sphere-to-rod transition of sodium dodecyl sulfate micelles on the dynamics of the membrane-bound tryptophan analogue, TOE, utilizing a combination of fluorescence spectroscopic approaches which include red edge excitation shift (REES). Our results show that REES and fluorescence spectroscopic parameters such as lifetime, anisotropy and acrylamide quenching of micelle-bound TOE are sensitive to the change in micellar organization accompanied by the sphere-to-rod transition.

Dynamics of a membrane-bound tryptophan analog in environments of varying hydration: a fluorescence approach

Tryptophan octyl ester (TOE) represents an important model for membrane-bound tryptophan residues. In this article, we have employed a combination of wavelength-selective fluorescence and time-resolved fluorescence spectroscopies to monitor the effect of varying degrees of hydration on the dynamics of TOE in reverse micellar environments formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. Our results show that TOE exhibits red edge excitation shift (REES) and other wavelength-selective fluorescence effects when bound to reverse micelles of AOT. Fluorescence parameters such as intensity, emission maximum, anisotropy, and lifetime of TOE in reverse micelles of AOT depend on [water]/[surfactant] molar ratio (w (o)). These results are relevant and potentially useful for analyzing dynamics of proteins or peptides bound to membranes or membrane-mimetic media under conditions of changing hydration.

Orientation and conformation of cell-penetrating peptide penetratin in phospholipid vesicle membranes determined by polarized-light spectroscopy.

The orientation and conformation of the cell-penetrating peptide "penetratin" associated with phospholipid vesicle membranes has been determined using polarized-light spectroscopy. The magnitude of orientation of penetratin is unprecedented for a solute in our membrane system, which we believe indicates a strong, specific interaction with the membrane. To validate the spectroscopic technique for studying the orientation of the two tryptophan residues of penetratin, we applied tryptophan octyl ester as a model compound. It is found to be incorporated in the membrane and preferentially oriented with its hydrophobic benzene edge of the indole chromophore pointing into the membrane and its hydrophilic groups oriented toward the water. For penetratin, the results indicate that a central alpha-helical part of the peptide is aligned parallel with the membrane surface, while the ends of the peptide adopt a planar structure. The planes of the two tryptophan side chains show a preferred orientation parallel with the membrane surface, indicating that they are not inserted into the membrane.

The polar headgroup of the detergent governs the accessibility to water of tryptophan octyl ester in host micelles

Many attempts have been made to rationalize the use of detergents for membrane protein studies [J. Biol. Chem. 264 (1989) 4907]. The barrier properties of the detergent headgroup may be one parameter critically involved in protein protection. In this paper, we analyzed these properties using a model system, by comparing the accessibility of tryptophan octyl ester (TOE) to water-soluble collisional quenchers (iodide and acrylamide) in three detergent micelles. The detergents used differed only in the chemical nature of their polar headgroups, zwitterionic for dodecylphosphocholine (DPC) and nonionic for octa(ethylene glycol) dodecyl monoether (C 12E 8) and dodecylmaltoside (DM). In all cases, in phosphate buffer at pH 7.5, the binding of 5 μM TOE was complete in the presence of a slight excess of detergent micelles over TOE molecules, resulting in a significant blue shift and greater intensity of TOE fluorescence emission. The resulting quantum yield of bound TOE was between 0.08 (in DPC) and 0.12 (in DM) with an emission maximum ( λ max) of ∼335 nm whatever the detergent micelle. Time-resolved fluorescence intensity decays of TOE at λ max were heterogeneous in all micelles (3–4 lifetime populations), with mean lifetimes of 1.7 ns in DPC, and 2 ns in both C 12E 8 and DM. TOE fluorescence quenching by iodide, in detergent micelles, yielded linear Stern–Volmer plots characteristic of a dynamic quenching process. The accessibility of TOE to this ion was the greatest with C 12E 8, followed by DPC and finally DM (Stern–Volmer quenching constants K sv of 2 to 5.5 M −1). In contrast, the accessibility of TOE to acrylamide was greatest with DPC, followed by C 12E 8 and finally DM ( K sv=2.7–7.1 M −1). TOE also presents less rotational mobility in DM than in the other two detergents, as shown from anisotropy decay measurements. These results, together with previous TOE quenching measurements with brominated detergents [Biophys. J. 77 (1999) 3071] provide reference data for analyzing Trp characteristics in peptide (and more indirectly protein)–detergent complexes. The main finding of this study was that TOE was less accessible (to soluble quenchers) in DM than in DPC and C 12E 8, the cohesion of DM headgroup region being suggested to play a role in the ability of this detergent to protect function and stability of solubilized membrane proteins.

Tryptamine derivatives as non-competitive N-methyl- d-aspartate receptor blockers: studies using [ 3H]MK-801 binding in rat hippocampal membranes

Derivatives of phenylethylamine and tryptamine share structural features with the non-competitive N-methyl- d-aspartate (NMDA) receptor blockers phencyclidine, ketamine, and MK-801. Tryptamine and phenylethylamine inhibited the specific binding of [ 3H]MK-801 to rat hippocampal membranes with IC 50‘s 190 and 905 μM, respectively. The corresponding amino acids phenylalanine and tryptophan were inactive, their methyl esters, however, were slightly more potent than the amines. The methyl ester of the naturally occurring l-tryptophan was 12 times more potent than the methyl ester of the d-isomer, whereas the corresponding isomers derived from phenylalanine exhibited no stereoselectivity. The potency of tryptamine was increased by substitutions as, e.g. in 5-methyltryptamine (IC 50 12 μM) and tryptophan octylester (IC 50 5.2 μM). Compounds formed in vivo from l-tryptophan and a lipophilic counterpart may function as endogenous non-competitive NMDA receptor blockers.

Influence of Reverse Micellar Environments on the Fluorescence Emission Properties of Tryptophan Octyl Ester

A number of recent studies have presented perspectives on the hydrophobic fluorescence probe tryptophan octyl ester (TOE). This molecule has attracted notable attention as a suitable model for the natural fluorophore tryptophan, in case of membrane proteins. We report here, for the first time, the fluorescence emission behaviour of TOE in reverse micelles of aerosol-OT (AOT) in n-heptane, containing different amounts of water. Relevant studies in representative homogeneous solvent media are also included for comparison. The fluorescence emission parameters (especially emission maximum, relative intensity, and anisotropy) of TOE are found to exhibit significant variation upon changes in the water/surfactant molar ratio (w0) of the reverse micelles. Fluorescence decay studies on TOE which we have also performed, indicate biexponential decay kinetics in reverse micelles as well as in homogeneous solvent media. The implications of these findings are examined in relation to the potentialities of TOE as a novel fluorescence probe for membrane proteins present in water restricted environments prevailing at the interfaces of biomembranes (for which reverse micelles serve as ideal model systems).

Tryptophan Octyl Ester in Detergent Micelles of Dodecylmaltoside: Fluorescence Properties and Quenching by Brominated Detergent Analogs

The fluorescence properties of tryptophan octyl ester (TOE), a hydrophobic model of Trp in proteins, were investigated in various mixed micelles of dodecylmaltoside (DM) and 7,8-dibromododecyl β-maltoside (BrDM) or 10,11-dibromoundecanoyl β-maltoside (BrUM). This study focuses on the mechanism via which these brominated detergents quench the fluorescence of TOE in a micellar system. The experiments were performed at a pH at which TOE is uncharged and almost completely bound to detergent micelles. TOE binding was monitored by its enhanced fluorescence in pure DM micelles or its quenched fluorescence in pure BrUM or BrDM micelles. In DM/BrUM and DM/BrDM mixed micelles, the fluorescence intensity of TOE decreased, as a nonlinear function of the molar fraction of brominated detergent, to almost zero in pure brominated detergent. The indole moiety of TOE is therefore highly accessible to the bromine atoms located on the detergent alkyl chain because quenching by bromines occurs by direct contact with the fluorophore. TOE is simultaneously poorly accessible to iodide (I −), a water-soluble collisional quencher. TOE time-resolved fluorescence intensity decay is heterogeneous in pure DM micelles, with four lifetimes (from 0.2 to 4.4 ns) at the maximum emission wavelength. Such heterogeneity may arise from dipolar relaxation processes in a motionally restricted medium, as suggested by the time-dependent (nanoseconds) red shift (11 nm) of the TOE emission spectrum, and from the existence of various TOE conformations. Time-resolved quenching experiments for TOE in mixed micelles showed that the excited-state lifetime values decreased only slightly with increases in the proportion of BrDM or BrUM. In contrast, the relative amplitude of the component with the longest lifetime decreased significantly relative to that of the short-lived species. This is consistent with a mainly static mechanism for the quenching of TOE by brominated detergents. Molecular modeling of TOE (in vacuum and in water) suggested that the indole ring was stabilized by folding back upon the octyl chain, forming a hairpin conformation. Within micelles, the presence of such folded conformations, making it possible for the entire molecule to be located in the hydrophobic part of the micelle, is consistent with the results of fluorescence quenching experiments. TOE rotational correlation time values, in the nanosecond range, were consistent with a hindered rotation of the indole moiety and a rotation of the complete TOE molecule in the pure DM or mixed detergent micelles. These results, obtained with a simple micellar model system, provide a basis for the interpretation of fluorescence quenching by brominated detergents in more complex systems such as protein- or peptide-detergent complexes.

Spectroscopic studies of the interaction of Ca2+-ATPase-peptides with dodecyl maltoside and its brominated analog.

The transmembrane sector of sarcoplasmic reticulum Ca2+-ATPase comprises ten putative transmembrane spans (M1-M10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca2+ binding: peptide M6 (amino acid residues 785-810), peptide M7-L (amino acid residues 808-847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818-847) which contains a shorter version of loop 6-7 than M7-L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the alpha-helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7-L peptide, the L6-7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca2+-ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl beta-D-maltoside or quenching using the recently introduced brominated analog of n-dodecyl beta-D-maltoside: 7,8-dibromododecyl beta-maltoside [de Foresta, B., Legros, N., Plusquellec, D., le Maire, M. & Champeil, P. (1996) Eur J. Biochem. 241, 343-354]. Our results indicate that M7-L and M7-S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size-exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl beta-D-maltoside/7,8-dibromododecyl beta-maltoside, using tryptophan octyl ester and solubilized Ca2+-ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca2+-ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca2+-ATPase and the rather short alpha-helix span of M6 may lead to exposure of a significant part of the C-terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca2+ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site-directed mutagenesis, that this transmembrane segment should be essential in Ca2+ binding.

Ionization, partitioning, and dynamics of tryptophan octyl ester: implications for membrane-bound tryptophan residues

The presence of tryptophan residues as intrinsic fluorophores in most proteins makes them an obvious choice for fluorescence spectroscopic analyses of such proteins. Membrane proteins have been reported to have a significantly higher tryptophan content than soluble proteins. The role of tryptophan residues in the structure and function of membrane proteins has attracted a lot of attention. Tryptophan residues in membrane proteins and peptides are believed to be distributed asymmetrically toward the interfacial region. Tryptophan octyl ester (TOE) is an important model for membrane-bound tryptophan residues. We have characterized this molecule as a fluorescent membrane probe in terms of its ionization, partitioning, and motional characteristics in unilamellar vesicles of dioleoylphosphatidylcholine. The ionization property of this molecule in model membranes has been studied by utilizing its pH-dependent fluorescence characteristics. Analysis of pH-dependent fluorescence intensity and emission maximum shows that deprotonation of the alpha-amino group of TOE occurs with an apparent pKa of approximately 7.5 in the membrane. The fluorescence lifetime of membrane-bound TOE also shows pH dependence. The fluorescence lifetimes of TOE have been interpreted by using the rotamer model for the fluorescence decay of tryptophan. Membrane/water partition coefficients of TOE were measured in both its protonated and deprotonated forms. No appreciable difference was found in its partitioning behavior with ionization. Analysis of fluorescence polarization of TOE as a function of pH showed that there is a decrease in polarization with increasing pH, implying more rotational freedom on deprotonation. This is further supported by pH-dependent red edge excitation shift and the apparent rotational correlation time of membrane-bound TOE. TOE should prove useful in monitoring the organization and dynamics of tryptophan residues incorporated into membranes.

Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions

The fluorescence of a membrane-bound tryptophan derivative (tryptophan octyl ester, TOE) has been examined as a model for tryptophan fluorescence from proteins in membrane environments. The depth-dependent fluorescence quenching of TOE by brominated lipids was found to proceed via a dynamic mechanism with vertical fluctuations playing a central role in the process. The activation energy for the quenching was estimated to be 1.3 kcal/mole. The data were analyzed using the distribution analysis (DA) method, which extends the conventional parallax method to account more realistically for the transbilayer distributions of both probe and quencher and for possible variations in the probe's accessibility. DA provides a better fit than the parallax method to data collected with TOE in membranes formed of lipids brominated at either the 4,5, the 6,7, the 9,10, or the 11,12 positions of the sn-2 acyl chain. DA yields information on the fluorophore's most probable depth in the membrane, its conformational heterogeneity, and its accessibility to the lipid phase. Previously reported data on cytochrome b5 and melittin were reanalyzed together with data obtained with TOE. This new analysis demonstrates conformational heterogeneity in melittin and provides estimates of the freedom of motion and exposure to the lipid phase of membrane-embedded tryptophans of cytochrome b5.

Distribution analysis of membrane penetration of proteins by depth-dependent fluorescence quenching

A new approach is presented to evaluate the depth-dependent quenching of the fluorescence of membrane-bound probes and integral proteins. By utilizing at least three quenchers of known and distinctly different depths, the following parameters can be recovered: most probable depth of the probe; dispersion of the depth distribution, which will depend on the size of probe and fluctuations in its position; and quenching efficiency, which is related to the exposure of a particular fluorophore to the lipid phase. The exposure of tryptophan residues in integral proteins can be quantitatively determined with respect to the model compound (tryptophan octyl ester). The proposed method was applied to the investigation of membrane complexes of the bee venom melittin and cytochrome b5.

Fluorescence quenching in model membranes: phospholipid acyl chain distributions around small fluorophores

Fluorescence quenching in lipid bilayers is treated by a new approach based on calculation of the probability distribution of quenching and nonquenching acyl chains around a fluorophore. The effect of acyl lattice site dependence (i.e., correlations of phospholipid sister chain occupancy of neighbor sites) was modeled by use of Monte Carlo simulations of acyl chain occupancy. This explicit accounting of site occupancy correlation was found to fit observed quenching behavior better than did a model wherein phospholipid quenchers are considered to be independent. A key aspect of this approach is to evaluate the rate for quenching in a bilayer composed of pure quenching lipid. In order to evaluate this quenching rate, and also to provide a strong test of the calculated probability distributions, we synthesized lipids with both acyl chains labeled with a quenching moiety (Br or nitroxide), as well as the more usual single-chain quenchers. The fluorescence of tryptophan octyl ester (TOE), and of the 1,6-diphenyl-1,3,5-hexatriene (DPH) derivatives trimethylammonium-DPH (TMA-DPH) and 1-lauroyl-2-(DPH-propionyl)phosphatidylcholine (DPH-PC), was examined. We obtained consistent results with all the fluorophores and quenchers indicating that up to 18 neighboring acyl sites can contribute to quenching, corresponding to two shells of acyl sites on a hexagonal lattice. Calculated discrete distributions of fluorescence intensities were converted into fluorescence lifetimes and compared with Gaussian and Lorentzian continuous lifetime distributions. The fluorescence quenching theory presented here may be used to explain quantitatively the heterogeneity of fluorophore environments in multicomponent membranes.

Esterase dependence of human neutrophil functions: lysozyme secretion, spontaneous migration, and continuity of the respiratory burst.

To assess the esterase dependency of human neutrophil secretion of lysosomal enzyme, the influence of several aromatic amino acid esters on phorbol myristate acetate-induced release of lysozome was studied. This function was found to be esterase-dependent on the basis of the inhibitory influence of such esters. Of the esters tested, tryptophan octyl ester, tryptophan benzyl ester, benzoyl tyrosine ethyl ester, and tryosine methyl ester were the most potent inhibitors of this response, being effective in the order listed. All esters with inhibitory effects were active at concentrations of less than or equal to 100 muM. Other neutrophil functions found to be similarly sensitive to the presence of these esters were spontaneous and chemotactic migration an continuity of the respiratory burst. The common inhibition profile of these neutrophil activities by the esters used suggests the activity of a common enzyme in these various functions.