Addition Of Trifluoroethanol Research Articles

Molecular dynamics simulation study of association in trifluoroethanol/water mixtures

Mixtures of Trifluoroethanol (TFE) and water with different proportions are studied using molecular dynamics simulations. The radial and spatial distribution functions, as well as the size distribution of TFE clusters are obtained from the trajectories. The variation of radial and spatial distribution functions with composition show that the addition of TFE enhances the water structure, but the hydrogen bonds between TFE molecules are broken as TFE is diluted with water. The TFE-rich solutions have stronger TFE-water hydrogen bonds. The clustering of TFE molecules in low concentration region is attributed to the hydrophobic interactions between CF(3) groups. The distribution of cluster sizes in solution supports these conclusions.

Protein stability modulated by a conformational effector: effects of trifluoroethanol on bovine serum albumin

The link between the thermodynamic properties of a solution and the conformational space explored by a protein is of fundamental importance to understand and control solubility, misfolding and aggregation processes. Here, we study the thermodynamic and conformational stability of a model protein, bovine serum albumin (BSA), by addition of trifluoroethanol (TFE), which is known to affect both the solvent properties and the protein structure. The solvent-mediated pair-wise interactions are investigated by static and dynamic light scattering, and by small angle X-ray scattering. The protein conformational details are studied by far- and near-UV circular dichroism (CD), and steady state fluorescence from tryptophan and from 1-anilino-8-naphthalene sulfonate (ANS). At low TFE concentrations, our results show that protein-protein interaction is dominated by steric repulsion accompanied by a consistent protein solvation. Minor local conformational changes also occur, but they do not affect the stability of BSA. At TFE concentrations above the threshold of 16% v/v, attractive interactions become prevalent, along with conformational changes related to a loosening of BSA tertiary structure. The onset of thermodynamic instability is triggered by the enhancement of hydrophobic attraction over repulsion, due to minor local changes of protein conformation and hydration. In the present context, TFE acts as a conformational effector, since it affects the intermolecular interaction and the activity of the proteins in solution through a direct mechanism.

Activity and Structural Changes of Euphorbia characias Peroxidase in the Presence of Trifluoroethanol

Activity assays, conformational changes and transitional switches between secondary structures of a peroxidase from Euphorbia characias were studied in the presence of trifluoroethanol and in the presence or absence of calcium ions. The addition of trifluoroethanol up to 10-20% first induced a drastic decrease of alpha-helix content followed by an increase of tryptophan fluorescence emission intensity, a progressive re-induction of the formation of alpha-helical elements concomitant with loss of enzyme activity. In the presence of calcium ions, the fluorescence of the enzyme almost remained unchanged in the trifluoroethanol concentration range 5-20%. Further increase in trifluoroethanol concentration led to a protein structure characterized by a progressive re-induction of alpha-helical elements, a remarkable increase of the tryptophan fluorescence and a loss of enzyme activity. These results indicate that calcium ions in Euphorbia peroxidase play an essential role in maintaining the hydrophobic interactions on the protein structure preserving enzymatic activity.

Conformational Changes of α-Chymotrypsin in a Fibrillation-Promoting Condition: A Molecular Dynamics Study

Amyloid nanofibril formation appears to be a generic property of polypeptide chains. α-Chymotrypsin (aCT) was recently driven toward amyloid-like aggregation by the addition of trifluoroethanol (TFE) at intermediate concentrations. In this study we employed a molecular dynamics simulation to investigate the early events in TFE-induced conformational changes of aCT that precede amyloid formation, and compared the results of the simulation with previous experiments. TFE molecules were found to rapidly replace the water molecules closely associated with the protein surface. The gyration radius, together with total and hydrophobic solvent-accessible surface areas of aCT, was significantly increased. In accord with the experimental observations, the extended β-conformation of backbone was increased. The secondary structural elements of aCT in water and TFE/water mixture showed a reasonable fit, whereas significant deviations were observed for several loops. These alterations originated largely from main-chain rotations at glycine residues. The catalytic active site and S1 binding pocket of the enzyme were also distorted in the TFE/water mixture. The obtained results are suggested to provide more insights into the conformational properties of the amyloid aggregation-prone protein species. Possible mechanisms of TFE-induced alterations in the conformation and dynamics of the protein structure are also discussed.

Synthesis of heterotactic and syndiotactic polyacrylamides via stereospecific radical polymerization of N‐tert‐butoxycarbonylacrylamide in the presence of fluorinated alcohols

Radical polymerization of a new protected monomer, N-tert-butoxycarbonylacrylamide, in toluene at −40 or 0 °C in the presence of fluorinated alcohols was investigated. The obtained polymers were successfully converted into polyacrylamide under acidic conditions. Further, it was revealed that atactic, heterotactic, and syndiotactic polymers were obtained with the addition of trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol, and nonafluoro-tert-butanol, respectively. To the best of our knowledge, these are the first syntheses of heterotactic and syndiotactic polyacrylamides.

Structure‐reactivity relationships for β‐galactosidase (Escherichia coli, lac Z): a second derivative effect on βnuc for addition of alkyl alcohols to an oxocarbenium ion reaction intermediate

Velocities for the synthesis of trifluoroethyl 2-deoxy-β-D-galactopyranoside by transfer of the 2-deoxygalactosyl group from β-galactosidase to trifluoroethanol were determined from studies of the β-galactosidase-catalyzed cleavage of 4-nitrophenyl-2-deoxy-β-D-galactopyranoside as the difference in rates of appearance of 4-nitrophenoxide anion and 2-D-deoxygalactose. These data were used to calculate a rate constant ratio of k(ROH)/k(s) = 2.3 M(-1) for partitioning of the intermediate between addition of trifluoroethanol and solvent water. Velocities for the synthesis of other alkyl 2-deoxy-β-D-galactopyranosides by transfer of the 2-deoxygalactosyl group from β-galactosidase to alkyl alcohols were determined from the effect of alkyl alcohols on the velocity of β-galactosidase-catalyzed cleavage of 4-nitrophenyl-2-deoxy-β-D-galactopyranoside in a reaction where breakdown of the intermediate is rate determining. These data were used to calculate rate constant ratios k(ROH)/k(s) for the reactions of eight alkyl alcohols. Absolute rate constants k(ROH) (M(-1) s(-1)) were calculated from k(ROH)/k(s) and k(s) = 0.002 s(-1) for the addition of water. A Brønsted coefficient of β(nuc) = -0.07 ± 0.08 was determined as the slope of a logarithmic correlation of k(ROH) against alcohol pK(a). The change from a 2-OH to a 2-H substituent at the β-D-galactopyranosyl intermediate causes a 0.12 ± 0.04 increase in the value of β(nuc) for alcohol addition. This anti-Hammond effect provides evidence that general basecatalyzed addition of alcohols to an enzyme bound β-D-galactopyranosyl oxocarbenium ion intermediate proceeds along a reaction coordinate in which there is strong coupling between carbon-oxygen bond formation and proton transfer from the alcohol to a basic residue at the enzyme.

Characterization of the structure and dynamics of mastoparan‐X during folding in aqueous TFE by CD and NMR spectroscopy

Mastoparan-X, a 14-residue peptide found in wasp venom, does not adopt a well-defined structure in water, but it folds into an alpha-helix upon addition of trifluoroethanol (TFE). At low levels of TFE, the peptide is partially folded, passing through intermediate stages of folding as the amount of TFE is increased. These partially folded states have been characterized by CD and NMR spectroscopy, and methods to estimate the helical content from CD, chemical shift, and nuclear overhauser effect (NOE) data are compared. Variation in the sign and intensity of NOE cross-peaks is observed in different regions of the peptide, indicative of greater mobility of the sidechains compared to the backbone of the peptide. Furthermore, variation in the sidechain mobility is observed, both between sidechains of different amino acids and within the sidechain of a given amino acid. By monitoring chemical shifts and NOE intensities as the TFE concentration is increased, the initiation site for helix formation could be identified. Furthermore, details of the peptide structure and dynamics during the folding process were elucidated.

The Effect of Trifluoroethoxy Groups on the Structures of Eight-Membered Ring-Containing Cyclic Phosphites1

New phosphites 1–4 were synthesized from phosphorus trichloride and an appropriate diphenol followed by the addition of trifluoroethanol in the presence of triethylamine. These phosphites are to serve as precursors in the syntheses of biorelated hypervalent phosphoranes. 1H, 19F, and 31P NMR spectra were recorded. X-ray analysis of 3 and 4 revealed that the sulfur-containing eight-membered ring was in a syn conformation that a allowed a sulfur donor interaction to the phosphorus atom, whereas for phosphite 2, the eight membered sulfonyl containing ring was in an anti conformation that did not allow a donor interaction to phosphorus from the oxygen atom of the sulfonyl group. Structural comparisons are made with related cyclic phosphites and phosphates having donor atoms in eight-membered rings.

Regulation of Catalytic Activity of Peptide–Heme Conjugate by Conformational Change with Trifluoroethanol

Abstract A peptide–heme conjugate in which two α-helix segments were covalently coupled at the heme propionates was designed, and the relationship between the structure and the peroxidase-like catalytic activity was examined. The activity and coordination state of heme were regulated by the conformational change of peptide induced upon the addition of trifluoroethanol.

Study of conformational properties of a biologically active peptide of fibronectin by circular dichroism, NMR and molecular dynamics simulation

Circular dichroism (CD), and NMR spectra have been recorded and molecular dynamics (MD) simulations have been performed in water and water-trifluoroethanol (TFE) mixed solvent for a synthetic biologically active 13-amino-acid fragment of human fibronectin and two related peptides. The CD results are interpreted on the basis of statistical analyses of MD trajectories and of ensuing calculations of CD spectra based on Schellman's matrix method. It is observed that the peptide conformation is quite variable in water and loses its mobility with the addition of TFE. (1)H-NOE data were found to be consistent with the most abundant calculated conformation.

2P105 Structural polymorphism of β_2-microglobulin amyloid fibrils induced by the addition of trifluoroethanol(31. Protein folding and misfolding (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P105 Structural polymorphism of β_2-microglobulin amyloid fibrils induced by the addition of trifluoroethanol(31. Protein folding and misfolding (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Synthesis and secondary structure in membranes of the Bcl‐2 anti‐apoptotic domain BH4

Solid phase synthesis of BH4, the 26 amino-acid domain (6RTGYDNREIVMKYIHYKLSQRGYEWD31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant beta-sheeted structures in water that convert progressively into alpha-helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes.

Trifluoroethanol-Induced Unfolding of Concanavalin A: Equilibrium and Time-Resolved Optical Spectroscopic Studies

Conformational structure changes in concanavalin A (Con A), a legume lectin protein which is composed of 18 beta-strands, induced by dissolving in 50% trifluoroethanol (TFE) were monitored at neutral and low pH by far- and near-UV circular dichroism (CD), fluorescence, and FTIR under equilibrium conditions. Stopped-flow studies using CD and fluorescence as well as FTIR, at low and high protein concentration, respectively, were carried out to follow the time-dependent conformation changes occurring after rapid mixing of the protein with TFE. Equilibrium CD results show that, upon addition of TFE, low-concentration Con A transforms to a highly alpha-helical conformation at both neutral and low pH. However, at neutral pH under high protein concentration conditions, aggregation and precipitation are eventually detected with FTIR, indicating that a final beta-structure is attained. Stopped-flow fluorescence shows the existence of an unfolding intermediate for pH 2.0 and 4.5, which could be related to the dissociation of the dimer form. However, evidence for an intermediate is not obtained at pH 6.7, where the native protein is a tetramer. Stopped-flow FTIR is consistent with these results, indicating formation of a H(+)-stabilized intermediate alpha-helical conformation before aggregation develops. Con A in TFE provides an example of an intermediate with non-native secondary structure appearing on the unfolding pathway. On the basis of the kinetic results obtained, an unfolding mechanism is proposed and some stable intermediates are identified. In turn, the slow structural change of Con A induced by TFE provides a useful model system for study of protein unfolding due to its accessibility with several spectroscopic and kinetic tools.

Conformational Prerequisites for Formation of Amyloid Fibrils from Histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.

Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins.

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.

Structural changes and facilitated association of tropoelastin

Circular dichroism studies of tropoelastin secondary structure show 4±1% α-helix in aqueous solutions. This is in contrast to the substantially higher amounts (up to 23±7%) of α-helix predicted by computer algorithms, which propose that regions of α-helix are limited to the alanine-rich cross-linking domains. Through the addition of trifluoroethanol, the amount of α-helix increased to 17±1%, equivalent to that expected on the basis of primary structure. The physiological ability of the protein to coacervate and the critical concentration of monomer required for coacervation were unaffected by levels of α-helix. However, the temperature required for coacervation decreased linearly with increasing α-helical structure, which correlates with the participation of α-helices in association. We propose that the alanine-rich cross-linking domains exist as nascent helices in tropoelastin in aqueous solution. We further suggest a novel mechanism for coacervation whereby formation of α-helices and subsequent helical side chain interactions limit the conformational flexibility of the polypeptide, to facilitate associations between hydrophobic domains during elastogenesis.

Conformational Switching and Fibrillogenesis in the Amyloidogenic Fragment of Apolipoprotein A-I

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.

The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins.

NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2. ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter P(parDE) and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE. Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and (3)J(Halpha-NH) scalar coupling constants showed that the N-terminal domain of ParD consists of a short beta-ribbon followed by three alpha-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors. (15)N longitudinal (T(1)) and transverse (T(2)) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain. An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein. This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins.

The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins

NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2. ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter PparDE and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE. Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and 3JHα-NH scalar coupling constants showed that the N-terminal domain of ParD consists of a short β-ribbon followed by three α-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors. 15N longitudinal (T1) and transverse (T2) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain. An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein. This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins.

Molten-globule structure and membrane binding of the N-terminal protease-resistant domain (63–193) of the steroidogenic acute regulatory protein (StAR)

The first step in steroidogenesis is the movement of cholesterol from the outer to inner mitochondrial membrane; this movement is facilitated by the steroidogenic acute regulatory protein (StAR). StAR has molten-globule properties at low pH and a protease-resistant N-terminal domain at pH4 and pH8 comprising residues 63–193. To explore the mechanism of action of StAR we investigated the structural properties of the bacterially expressed N-terminal domain (63–193 StAR) using CD, limited proteolysis and NMR. Far- and near-UV CD showed that the amount of secondary structure was greater at acidic than at neutral pH, but there was little tertiary structure at any pH. Unlike 63–193 StAR liberated from N-62 StAR by proteolysis, biosynthetic 63–193 StAR was no longer resistant to trypsin or proteinase K at pH7, or to pepsin at pH4. Addition of trifluoroethanol and SDS increased secondary structure at pH7, and dodecylphosphocholine and CHAPS increased secondary structure at pH2, pH4 and pH7. However, none of these conditions induced tertiary structure, as monitored by near-UV CD or NMR. Liposomes of phosphatidylcholine, phosphatidylserine and their mixture increased secondary structure of 63–193 StAR at pH7, as monitored by far-UV CD, and stable protein–liposome complexes were identified by gel-permeation chromatography. These results provide further evidence that the N-terminal domain of StAR is a molten globule, and provide evidence that this conformation facilitates the interaction of the N-terminal domain of StAR with membranes. We suggest that this interaction is the key to understanding the mechanism of StAR's action.