1-palmitoyl-2-oleoylphosphatidylcholine and tryptophan-octyl-ester

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.

Topics: Lipid Bilayers; Membrane Proteins; Molecular Dynamics Simulation; Phosphatidylcholines; Spectrometry, Fluorescence; Tryptophan

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.

Topics: Carrier Proteins; Cell Membrane Permeability; Cell-Penetrating Peptides; Light; Liposomes; Models, Molecular; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids; Protein Conformation; Spectrum Analysis; Tryptophan

The partitioning of native cytochrome b5 and a mutant form, where Trp-108 and Trp-112 were both replaced by Leu, into small unilamellar lipid vesicles was examined. The vesicles were made from phosphatidylcholines containing mono- and di-unsaturated acyl chains. As these amphipathic proteins self-associate in aqueous solution, the binding was not monitored by a simple lipid titration experiment but by an exchange assay using fluorescence quenching by brominated lipids. Each protein had a greater affinity for lipids containing mono-unsaturated chains than for vesicles containing di-unsaturated chains, and the affinities of both proteins increased in buffers of higher ionic strength. The native protein had a higher affinity than the mutant protein for all vesicles; the ratio of the affinities was relatively constant at approximately 30. This corresponds to a difference in the free energy of partitioning of 2 kcal mol(-)(1). The fluorescence quantum yields of both proteins were much lower in lipids with di-unsaturated chains whereas a similar lowering was not seen with a simple Trp compound. These data suggest that the decreased membrane hydrophobicity seen by the proteins in di-unsaturated membranes is not an inherent property of the bilayer but is induced by the insertion of the protein. Further, the similar behavior of the two proteins suggests this modulation is not sensitive to the amino acid side chains of the inserted domain.

Topics: Bromine; Chromatography, Gel; Cytochromes b5; Escherichia coli; Fluorescent Dyes; Lipid Bilayers; Membrane Proteins; Mutagenesis, Site-Directed; Osmolar Concentration; Phosphatidylcholines; Protein Binding; Spectrometry, Fluorescence; Tryptophan

Fluorescence quenching is used to gain information on the exposure of tryptophan residues to lipid in membrane-bound proteins and peptides. A protocol is developed to calculate this exposure, based on a comparison of quenching efficiency and of a fluorescence lifetime (or quantum yield) measured for a protein and for a model tryptophan-containing compound. Various methods of analysis of depth-dependent quenching are compared and three universal measures of quenching profile are derived. One of the measures, related to the area under profile, is used to estimate quenching efficiency. The method is applied to single tryptophan mutants of a membrane-anchoring nonpolar peptide of cytochrome b(5) and of an outer membrane protein A. Analysis of quenching of the cytochrome's nonpolar peptide by a set of four brominated lipids reveals a temperature-controlled reversible conformational change, resulting in increased exposure of tryptophan to lipid and delocalization of its transverse position. Kinetic quenching profiles and fluorescence binding kinetics reported by Kleinschmidt et al. (Biochemistry (1999) 38, 5006-5016) were analyzed to extract information on the relative exposure of tryptophan residues during folding of an outer membrane protein A. Trp-102, which translocates across the bilayer, was found to be noticeably shielded from the lipid environment throughout the folding event compared to Trp-7, which remains on the cis side. The approach described here provides a new tool for studies of low-resolution structure and conformational transitions in membrane proteins and peptides.

Topics: Bacterial Outer Membrane Proteins; Cytochromes b5; Fluorescent Dyes; In Vitro Techniques; Kinetics; Membrane Lipids; Membrane Proteins; Models, Chemical; Peptides; Phosphatidylcholines; Protein Folding; Spectrometry, Fluorescence; Tryptophan