melitten and tryptophan-octyl-ester

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 pr

Topics: Hydrogen Bonding; Melitten; Membrane Proteins; Solvents; Spectrometry, Fluorescence; Spectrum Analysis, Raman; Tryptophan

Determination of the topology of peptides in membranes is important for characterizing and understanding the interactions of peptides with membranes. We describe a method that uses fluorescence quenching arising from resonance energy transfer ("FRET") for determining the topology of the tryptophan residues of peptides partitioned into phospholipid bilayer vesicles. This is accomplished through the use of a novel lyso-phospholipid quencher (lysoMC), N-(7-hydroxyl-4-methylcoumarin-3-acetyl)-1-palmitoyl-2-hydroxy-sn-gly cero-3-phosphoethanolamine. The design principle was to anchor the methylcoumarin quencher in the membrane interface by attaching it to the headgroup of lyso-phosphoethanolamine. We show that lysoMC can be incorporated readily into large unilamellar phospholipid vesicles to yield either symmetrically (both leaflets) or asymmetrically (outer leaflet only) labeled bilayers. LysoMC quenches the fluorescence of membrane-bound tryptophan by the Förster mechanism with an apparent R(0) that is comparable to the thickness of the hydrocarbon core of a lipid bilayer (approximately 25 A). Consequently, the methylcoumarin acceptor predominantly quenches tryptophans that reside in the same monolayer as the probe. The topology of a peptide's tryptophan in membranes can be determined by comparing the quenching in symmetric and asymmetric lysoMC-labeled vesicles. Because it is essential to know that asymmetrically incorporated lysoMC remains so under all conditions, we also developed a second type of FRET experiment for assessing the rate of transbilayer diffusion (flip-flop) of lysoMC. Except in the presence of pore-forming peptides, there was no measurable flip-flop of lysoMC, indicating that asymmetric distributions of quencher are stable. We used these methods to show that N-acetyl-tryptophan-octylamide and tryptophan-octylester rapidly equilibrate across phosphatidylcholine (POPC) and phosphatidylglycerol (POPG) bilayers, while four amphipathic model peptides remain exclusively on the outer monolayer. The topology of the amphipathic peptide melittin bound to POPC could not be determined because it induced rapid flip-flop of lysoMC. Interestingly, melittin did not induce lysoMC flip-flop in POPG vesicles and was found to remain stably on the external monolayer.

Topics: Amino Acid Sequence; Coumarins; Energy Transfer; Fluorescent Dyes; Lipid Bilayers; Lysophospholipids; Melitten; Molecular Sequence Data; Peptides; Phosphatidylethanolamines; Spectrometry, Fluorescence; Structure-Activity Relationship; Tryptophan