Subject(s)
Benzodiazepinones/pharmacology , Enzyme Inhibitors/pharmacology , Thiolester Hydrolases/antagonists & inhibitors , Animals , Benzodiazepinones/chemical synthesis , Benzodiazepinones/chemistry , Cell Differentiation/drug effects , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , In Vitro Techniques , Molecular Structure , PC12 Cells , Rats , Structure-Activity Relationship , Thiolester Hydrolases/chemistry , Time Factors , ras Proteins/chemical synthesis , ras Proteins/chemistry , ras Proteins/drug effectsABSTRACT
The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity of the lipidated peptide to a fluid, DMPC-rich environment. This phase separation leads to the formation of peptide-containing domains with high fluorescence intensity that become progressively smaller with decreasing temperature. As a result, the local concentration of the peptide increases steadily within the confines of the shrinking domains. At the lowest temperatures, where the acyl-chain order parameter of the membrane has already drastically increased and the membrane achieves a liquid-ordered character, an efficient lipid sorting mechanism is no longer supported and aggregation of the peptide into small clusters prevails. We can conclude that palmitoyl/farnesyl dual-lipidated peptides do not associate with liquid-ordered or gel-like domains in phase-separated bilayer membranes. In particular, the study shows the interesting ability of the peptide to induce formation of fluid microdomains at physiologically relevant cholesterol concentrations, and this effect very much depends on the concentration of fluid vs ordered lipid molecules.
Subject(s)
Lipids/chemistry , Lipoproteins/chemistry , Oligopeptides/chemistry , Cholesterol/chemistry , Microscopy, Fluorescence/methods , Models, Chemical , Molecular Structure , Phosphatidylcholines/chemistry , Protein Binding , TemperatureABSTRACT
The temperature-dependent behavior of a solvated oligopeptide, GVG(VPGVG), is investigated. Spectroscopic measurements, thermodynamic measurements, and molecular dynamics simulations find that this elastinlike octapeptide behaves as a two-state system that undergoes an "inverse temperature" folding transition and reentrant unfolding close to the boiling point of water. A molecular picture of these processes is presented, emphasizing changes in the dynamics of hydrogen bonding at the protein/water interface and peptide backbone librational entropy.
Subject(s)
Elastin/chemistry , Oligopeptides/chemistry , Hydrogen Bonding , Protein Folding , Spectroscopy, Fourier Transform Infrared , Temperature , ThermodynamicsABSTRACT
Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68 % yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.
Subject(s)
Lipids/chemistry , Lipoproteins/chemical synthesis , Peptides/chemistry , Acids/chemistry , Alkalies/chemistry , Amino Acid Sequence , Fluorescent Dyes/chemistry , Lipids/chemical synthesis , Maleimides/chemistry , Peptides/chemical synthesisABSTRACT
Characteristic partial structures of lipidated proteins embodying different lipid groups as well as additional fluorescent tags or a maleimide for coupling to proteins can be synthesized readily by means of a new solid-phase technique employing the oxidative cleavage of the hydrazide linker as well as on-resin farnesylation and palmitoylation after appropriate deprotection of cysteine thiol groups as the key steps.
