Trifluoroethanol

The structural stabilizing property of 2,2,2-trifluoroethanol (TFE) in peptides has been widely demon strated. More recently, TFE has been shown to enhance secondary structure content in globular proteins, and to influence quaternary interactions in protein multimers. The molecular mechanisms by which TFE exerts its influence on peptide and protein structures remain poorly understood. The present analysis integrates the known physical properties of TFE with a variety of experimental observations on the interaction of TFE with peptides and proteins and on the properties of fluorocarbons. Two features of TFE, namely the hydrophobicity of the trifluoromethyl group and the hydrogen bonding character (strong donor and poor acceptor), emerge as the most important factors for rationalising the observed effects of TFE. A model is proposed for TFE interaction with peptides which involves an initial replacement of the hydration shell by fluoroalcohol molecules, a process driven by apolar interactions and favourable entropy of dehydration. Subsequent bifurcated hydrogen-bond formation with peptide carbonyl groups, which leave intramolecular interactions unaffected, promotes secondary structure formation. © Munksgaard 1996.

Reversed-phase high-pressure liquid chromatography analysis and purification of three hydrophobic, aggregation-prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2-trifluoroethanol (TFE). The addition of 10-16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE-containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711: 181-186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM-peptide purification. Characterization of the potential side reactions using MALDI and ESI-LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE-formic acid possibly induces O-formylation of the Ser residue and N-formylation of the N-t...

Magainin2 is a 23-residue antibiotic peptide that disrupts the ionic gradient across certain cell membranes. Two-dimensional 1H NMR spectroscopy was used to investigate the structure of the peptide in three of the membrane environments most commonly employed in biophysical studies. Sequence-specific resonance assignments were determined for the peptide in perdeuterated dodecylphosphocholine (DPC) and sodium dodecylsulfate micelles and confirmed for the peptide in 2,2,2-trifluoroethanol solution. The secondary structure is shown to be helical in all of the solvent systems. The NMR data were used as a set of restraints for a simulated annealing protocol that generated a family of three-dimensional structures of the peptide in DPC micelles, which superimposed best between residues 4 and 20. For these residues, the mean pairwise rms difference for the backbone atoms is 0.47 +/- 0.10 A from the average structure. The calculated peptide structures appear to be curved, with the bend center...

A biodegradable magnesium matrix and beta-tricalcium phosphate (β-TCP) particles reinforced composite Mg-2Zn-0.5Ca/1beta-TCP (wt.%) was fabricated for biomedical applications by the novel route of combined high shear solidification (HSS) and equal channel angular extrusion (ECAE). The as-cast composite obtained by HSS showed a fine and equiaxed grain structure with globally uniformly distributed β-TCP particles in aggregates of 2-25μm in size. The ECAE processing at 300°C resulted in further microstructural refinement and the improvement of β-TCP particle distribution. During ECAE, the β-TCP aggregates were broken into smaller ones or individual particles, forming a dispersion in the matrix. Such fabricated composite exhibited enhanced hardness and in vitro corrosion resistance. The enhanced hardness was attributed to both the addition of β-TCP particles and grain refinement while the development of a Ca-P rich surface layer from β-TCP during corrosion was responsible for the improv...

Viperin, an antiviral protein, has been shown to contain a CX(3)CX(2)C motif, which is conserved in the radical S-adenosyl-methionine (SAM) enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a low-resolution predicted structure. The results show that Viperin is an α -β protein containing iron-sulfur cluster at the center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. Conformational...

Conformational and functional changes of cardosin A, an aspartic protease of vegetal origin, in the presence of 2,2,2-trifluoroethanol (TFE), were assessed. TFE induced alterations of cardosin activity and conformation that differed with the solvent concentration. MD simulations showed that there are significant local alterations in protein flexibility and TFE molecules were found to replace several hydration molecules in the active site of the enzyme. This may explain some of the activity loss observed in the presence of TFE, especially at low TFE concentrations, as well as the recovery of enzyme activity upon aqueous dilution, indicating the release of the TFE molecules from the active site.

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH(2)), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO(-)) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO(-), a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH(2) and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO(-) leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH(2) exists mainly as a rigid structure, EMP-AF-COO(-) presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.