TFE Solutions Research Articles

CH3–X Reductive Elimination Reactivity of PtIVMe Complexes Supported by a Sulfonated CNN Pincer Ligand (X = OH, CF3CO2, PhNMe2+)

Three new dihydrocarbyl LPtIVMe(Y) complexes (6–8; Y = Cl, I, OCH2CF3) supported by the sulfonated CNN pincer ligand L have been prepared and characterized. The reaction of these complexes with a number of nucleophiles (H2O, CF3CO2–, Me2SO, PhNMe2) resulting in the formation of corresponding C–X coupled products CH3–X has been studied in 2,2,2-trifluoroethanol (TFE) and DMSO. In TFE or DMSO solutions at 22 °C, in the presence of PhNMe2, a quantitative formation of a C–N coupled product, PhNMe3+, was observed for 6–8, with the reactivity decreasing in the order 6 > 7 > 8. The use of NaO2CCF3 in TFE solutions was less efficient, leading to the production of MeO2CCF3 in 60% yield after 22 h at 70 °C. In DMSO the C–O coupling was high yielding when aqueous trifluoroacetic acid was used to produce methanol (87% after 3 h at 80 °C) or when NaO2CCF3 was used to form MeO2CCF3 (80% after 1.5 h at 80 °C), with Me3SO+ being a minor byproduct. The kinetics study of the reaction between 6–8 and PhNMe2 in TFE has revea...

Can 2,2,2-trifluoroethanol be an efficient protein denaturant than methanol and ethanol under thermal stress?

Monohydric alcohols, such as methanol (MEH), ethanol (ETH) and 2,2,2-trifluoroethanol (TFE), have significant effects on biological processes including the protein folding-unfolding phenomenon. Among the several monohydric alcohols, TFE, a fluorine-substituted alcohol, is known to induce a helical structure in proteins. In this work, we report the heterogeneous unfolding phenomenon of a small protein Chymotrypsin Inhibitor 2 in various concentrations of methanol, ethanol and TFE solutions by performing atomistic molecular dynamics simulation studies. Our study reveals that the unfolding phenomenon of CI2 under thermal stress majorly depends on the concentration and the nature of the alcohol. The presence of alcohols in general has been noted to accelerate the unfolding process compared to pure water and TFE, among them all, has been found to speed up the unfolding time scale at low concentrations. The molecular contact frequency between protein and alcohol follows the trend, MEH < ETH < TFE at low concentrations, whereas the trend becomes MEH ∼ ETH > TFE at more concentrated solutions. The differential water-mediated and self-clustering phenomena of alcohols, diverse protein-alcohol hydrogen bond strengths and the concentration dependent restricted inhomogeneous protein-water as well as protein-alcohol hydrogen bond dynamics suggest that TFE, a well known α-helix stabilizer, could be a good competitor among its class of denaturants.

C–H and O2 Activation at a Pt(II) Center Enabled by a Novel Sulfonated CNN Pincer Ligand

A novel sulfonated CNN pincer ligand has been designed to support CH and O2 activation at a Pt(II) center. The derived cycloplatinated aqua complex 7 was found to be one of the most active reported homogeneous Pt catalysts for H/D exchange between studied arenes (benzene, benzene-d6, toluene-d8, p-xylene, and mesitylene) and 2,2,2-trifluoroethanol (TFE) or 2,2,2-trifluoroethanol-d; the TON for C6D6 as a substrate is >250 after 48 h at 80 °C. The reaction is very selective; no benzylic CH bond activation was observed. The per-CH-bond reactivity diminishes in the series benzene (19) > toluene (p-CH:m-CH:o-CH = 1:0.9:0.2) > xylene (2.9) > mesitylene (1.1). The complex 7 reacts slowly in TFE solutions under ambient light but not in the dark with O2 to selectively produce a Pt(IV) trifluoroethoxo derivative. The H/D exchange reaction kinetics and results of the DFT study suggest that complex 7, and not its TFE derivatives, is the major species responsible for the arene CH bond activation. The reaction deuteriu...

Molecular dynamics simulation study of solvation effects of water and trifluoroethanol on gamma-aminobutyric acid (GABA)

Solvation structure and association of γ-aminobutyric acid (GABA) zwitterions in aqueous TFE solutions are investigated using molecular dynamics simulation and the AMBER03 force field. Radial distribution functions are used to discuss the effect of solvents on GABA and vice versa. It is shown that GABA causes structure making among water molecules through kosmotropic effect and structure breaking among water and TFE molecules through chaotropic effect. Moreover, the weighted histogram analysis method (WHAM) is used for the calculation of potential of mean force (PMF) from MD simulations. Using the extracted PMFs, the association free energies and the association equilibrium constants for GABA in TFE and in water are calculated. It is shown that the association of GABA molecules in TFE is more prospective than that in water and the probability of association increases with GABA concentration.

Clicked bis-PEG-peptide conjugates for studying calmodulin-Kv7.2 channel binding.

The recombinant Kv7.2 calmodulin (CaM) binding site (Q2AB CaMBD) shows a high tendency to aggregate, thus complicating biochemical and structural studies. To facilitate these studies we have conceived bis-PEG-peptide CaMBD-mimetics linking helices A and B in single, easy to handle molecules. Short PEG chains were selected as spacers between the two peptide molecules, and a Cu(i)-catalyzed cycloaddition (CuAAC) protocol was used to assemble the final bis-PEG-peptide conjugate, by the convenient functionalization of PEG arms with azide and alkyne groups. The resulting conjugates, with a certain helical character in TFE solutions (CD), showed nanomolar affinity in a fluorescence CaM binding in vitro assay, higher than just the sum of the precursor PEG-peptide affinities, thus validating our design. The approach to these first described examples of Kv7.2 CaMBD-mimetics could pave the way to chimeric conjugates merging helices A and B from different Kv7 subunits.

A study of alcohol and temperature effects on aggregation of β-lactoglobulin by viscosity and small-angle X-ray scattering measurements

Small-angle X-ray scattering (SAXS) of aqueous solutions of methanol and 2,2,2-trifluoroethanol (TFE) of β-lactoglobulin (β-Lg) in concentrations of 30 and 63mg/mL was measured as a function of alcohol concentration in a temperature range of 298–363K to investigate the effects of alcohol and temperature on aggregation of the protein. The viscosity of aqueous solutions of methanol, ethanol and TFE of β-Lg in a concentration of 2mg/mL was also measured as a function of alcohol concentration at an ambient temperature. The relative viscosity, η/η0, where η and η0 are the viscosities of the protein solutions and the solvents, respectively, showed a maximum at around 50%, 40% and 20% of the methanol, ethanol and TFE solutions, respectively. This finding suggests that the aggregation of β-Lg of a low concentration occurs at the specific alcohol concentrations. The SAXS data showed that the aggregation of β-Lg in terms of the aggregation number and the radius of gyration initiates at an alcohol concentration of ~40% for the methanol solutions and ~10% for the TFE solutions and increases with alcohol concentration at ambient temperature. Furthermore, the temperature of thermal denaturation of β-Lg decreased by ~30K for the TFE solution and by ~20K for the methanol solution, compared with the case for water (348K). Thus, the addition of TFE promoted the thermal denaturation of the protein more effectively than methanol. The aggregation structure of the thermally denatured protein was independent of type and concentration of alcohol in the concentrated protein solutions. Furthermore, the fractal dimension of the aggregates was ~2.6, indicating a three dimensional network structure. However, in the diluted protein solutions, the aggregate structure after the thermal denaturation was likely to be a low cross-linked network gel in the methanol solution, which was similar to that in water. On the other hand, more complex aggregates tended to form in the TFE solution.

The amyloidogenic SEVI precursor, PAP248-286, is highly unfolded in solution despite an underlying helical tendency

Amyloid fibers in human semen known as SEVI (semen-derived enhancer of viral infection) dramatically increase the infectivity of HIV and other enveloped viruses, which appears to be linked to the promotion of bridging interactions and the neutralization of electrostatic repulsion between the host and the viral cell membranes. The SEVI precursor PAP248–286 is mostly disordered when bound to detergent micelles, in contrast to the highly α-helical structures found for most amyloid proteins. To determine the origin of this difference, the structures of PAP248–286 were solved in aqueous solution and with 30% and 50% trifluoroethanol. In solution, pulsed field gradient (PFG)-NMR and 1H-1H NOESY experiments indicate that PAP248–286 is unfolded to an unusual degree for an amyloidogenic peptide but adopts significantly helical structures in TFE solutions. The clear differences between the structures of PAP248–286 in TFE and SDS indicate electrostatic interactions play a large role in the folding of the peptide, consistent with the slight degree of penetration of PAP248–286 into the hydrophobic core of the micelle. This is another noticeable difference between PAP248–286 and other amyloid peptides, which generally show penetration into at least the headgroup region of the bilayer, and may explain some of the unusual properties of SEVI.

Influence of N‐terminus modifications on the biological activity, membrane interaction, and secondary structure of the antimicrobial peptide hylin‐a1

Recently the peptide Hy-a1 (IFGAILPLALGALKNLIK), with antimicrobial activity, was isolated from the skin secretion of the frog Hypsiboas albopunctatus. The aim of the present work was to evaluate four analogues with introduction of acetyl group, Asp or Lys at the N-terminus of antimicrobial peptide Hy-al to supply information about the relationship of structure-biological activity. The antimicrobial activities were assayed by measuring growth inhibition of four species of bacteria and four species of fungus. The hemolytic activity was also tested. The peptide containing Trp instead of Leu in position 6 (for fluorescence studies) presented MIC values comparable to wild type sequence: 32 μmol L(-1) , 32 μmol L(-1) , 8 μmol L(-1) , and 2 μmol L(-1) for E. coli, P. aeruginosa, S. aureus, and B. subtilis, respectively. Two peptides with this modification and containing one acetyl group or Asp residue at the N-terminal region showed activities only against Gram-positive bacteria. Different results were observed when the residue added was Lys. In this case, the activity against the microorganisms was sustained or increased. Conformational properties were investigated by CD techniques in water, TFE, and in zwitterionic micelles (LPC). The CD experiments demonstrated that, in water, the peptides had a random structure, but in TFE and LPC solutions they acquired an ordered structure, composed mainly by α-helix. However, these data have no relationship with activity against Gram-positive bacteria. These results showed that the N-terminal region of the peptide Hy-a1 has key roles in its antibacterial action toward different types of bacteria.

Tandem Repeating Modular Proteins Avoid Aggregation in Single Molecule Force Spectroscopy Experiments

We have used single molecule force spectroscopy to explore the unfolding and refolding behavior of the immunoglobulin-like I27 protein in aqueous 2,2,2-trifluoroethanol (TFE). In bulk solution experiments, a 28% v/v TFE solution has previously been observed to enhance intermolecular attractions and lead to misfolding and aggregation of tandem modular proteins of high sequence identity. In our single molecule experiments, however, we measure successful refolding of the polyprotein I27(8) in all TFE solutions up to 35% v/v. Using a single molecule micromanipulation technique, we have shown that refolding of a polyprotein with identical repeats is not hindered by the presence of this cosolvent. These experimental results provide new insight into the properties of tandem repeating proteins and raise interesting questions as to the evolutionary success of such proteins in avoiding misfolding and aggregation.

NMR Solution Structure of Neurotensin in Membrane-Mimetic Environments: Molecular Basis for Neurotensin Receptor Recognition

Neurotensin (NT) is a 13-residue neuropeptide that exerts multiple biological functions in the central and peripheral nervous system. Little is known about the structure of this neuropeptide, and what is known only concerns its C-terminal part. We determined here for the first time the structure of the full-length NT in membrane-mimicking environments by means of classical proton-proton distance constraints derived from solution-state NMR spectroscopy. NT was found to have a structure at both its N and C termini, whereas the central region of NT remains highly flexible. In TFE and HFIP solutions, the NT C-terminus presents an extended slightly incurved structure, whereas in DPC it has a beta turn. The N-terminal region of NT possesses great adaptability and accessibility to the microenvironment in the three media studied. Altogether, our work demonstrates a structure of NT fully compatible with its NTR-bound state.

Solvatochromism in the aromatic ketone benzo[ b]fluorenone

Spectroscopic studies on benzo[ b]fluorenone (BF) solvatochromism in several pure aprotic and alcoholic solvents are reported. Fluorescence quenching was not observed in pure ethanol, unlike the related aromatic ketone 9-fluorenone (9-FL). Results in the more strongly hydrogen bonding alcohol 2,2,2-trifluoroethanol (TFE) demonstrate fluorescence quenching, a red shift in emission, and indicate the presence of at least two ground state species in BF–TFE solutions. These results suggest BF has less ability to hydrogen bond with ethanol than 9-FL; this appears to relate to the difference in aromaticity between these two compounds.

Demonstration of the Ring Conformation in Polyproline by the Raman Optical Activity

Raman and Raman optical activity (ROA) spectra of poly-L-proline were recorded in a wide frequency range and analyzed with respect to the proline side chain conformation. The analysis was based on comparison to ab initio simulations of spectral band positions and intensities. The presence of two conformer states of the five-member ring was found, approximately equally populated in the polypeptide. Additionally, Raman and ROA spectral shapes indicated that the peptide adopts the polyproline II helical conformation, in both aqueous and TFE solutions. The helix, however, is perturbed by fluctuations, which affects the vibrational coupling among amino acid residues and broadens the ROA bands. Contributions of the side and main peptide chains to the polyproline ROA intensities have comparable magnitudes. Thus understanding of the origins of both signals is important for determination of the peptide structure by ROA.

Local Chemical Potential Equalization Model for Cosolvent Effects on Biomolecular Equilibria

A simple model for describing the effects of cosolvents on biomolecular equilibria in solution is presented. The model is developed using the Kirkwood−Buff theory of solutions and relates changes in the chemical potential of the cosolvent, due to the presence of the solute, to changes in the cosolvent (and water) concentrations in the vicinity of the biomolecule. The model is then used to determine the dependence of changes in the free energy for protein denaturation on cosolvent concentration. The experimentally observed linearity for urea, and small deviation from linear behavior for guanidinium chloride denaturation, are reproduced. In addition, the model is also predicted to provide a good description of the helix-inducing effects of TFE solutions.

Solution conformational study of nociceptin and its 1–13 and 1–11 fragments using circular dichroism and two‐dimensional NMR in conjunction with theoretical conformational analysis

Conformational studies of nociceptin (NC-NH2), its fully active fragment, NC(1-13)-NH2, and two significantly less potent fragments, NC(1-13)-OH and NC(1-11)-OH, were conducted in water and TFE solutions by the employment of circular dichroism, and in DMSO-d6 by 2DNMR spectroscopy in conjunction with theoretical conformational analysis. The conformations of all thepeptides studied were calculated taking two approaches. The first assumes multiconformational equilibrium of the peptide studied, which is characterized by a set of conformations (and their statistical weight values)obtained from a global conformational analysis using three methods: the electrostatically driven Monte-Carlo (EDMC) with the ECEPP/3 force field, the simulated annealing (SA) protocols in the AMBER and CHARMM force fields. The second approach incorporates the interproton distance and dihedral angle constraints into the starting conformation. Calculations were performed using the distance geometry and SA protocol in the CHARMM force field implemented in the X-PLOR program. The CD experiments indicated that for the active peptides, hydrophobic solvents induced a significantly higher (compared with those remaining)content order, probably a helical structure. Unfortunately, as a result of the conformational flexibility of thepeptides, the analysis of conformations obtained with both approaches and different force fields did not alllow the selection of any structural elements of the NC peptides that might be connected with their bioactivity. The only common element found in most conformations of the active peptides was a helical character of fragment 8-13, which allowed the side chains of basic amino acid residues to be exposed to the outside of the molecule and probably to interact with the ORL1 receptor.

α and β Conformational preferences in fibril forming peptides characterised using NMR and CD techniques

Peptide fragments taken from residues 18‒54 of short consensus repeat 3 (SCR3) from the human complement receptor CR1 have been found in aqueous solution to slowly aggregate and form fibrils. NMR studies of the monomeric form of these peptides show that they are essentially unfolded in aqueous solution and that they all have an increased helicity in TFE solutions. The behaviour of residues 28‒31 from SCR3 is particularly interesting. These residues have a high β‐sheet propensity in the native protein and a seven peptide containing their sequence is found to form fibrils despite its short length. However, NMR studies show that these residues adopt a well‐defined α‐helix in 80% TFE and under these conditions fibril formation has not been observed. These data demonstrate the strong dependence of conformational propensities on environment.

NMR conformational analysis of antide, a potent antagonist of the gonadotropin releasing hormone.

Antide is a decapeptide [(N-Ac-D-Nal(1)-D-Cpa(2)-D-Pal(3)-Ser(4)-Lys(Nic)(5)-D-Lys(Nic)(6)-Leu(7)-Ilys(8)-Pro(9)-D-Ala(10)-NH(2)] that acts in vivo as an antagonist of GnRH (gonadotropin-releasing hormone). The conformational behavior of antide has been studied in water, TFE, DMF, and DMSO solutions by means of 2D-NMR spectroscopy and molecular dynamics calculations. Antide adopts in aqueous solution a delta-shaped backbone conformation, which is characterized by an irregular turn around residues D-Pal(3)-Ser(4) and by the close spatial proximity of the side chains belonging to D-Nal(1) and Ilys(8) (as many as 17 NOE peaks were detected between these side chains). The side-chain protons of Ilys(8) (especially the H(gamma) ones) present remarkably upfield shifted resonances, because of ring current effects induced by the naphthyl moiety. The upfield shifted resonances of the Ilys(8) H(gamma) hydrogen atoms are strictly characteristic of the water delta-shaped conformation and can be considered as structure markers. The observation of ring current shifted Ilys(8) H(gamma) resonances under different conditions (temperature, pH, solvent) indicates a remarkable stability of the water delta-shaped conformation. Such a conformation is at least partially disrupted in solvent mixtures containing high percentages of organic solvents. TFE can induce a well-defined conformation, which is characterized by an S-shaped backbone conformation. In DMF and DMSO solution, the molecule is basically endowed with a random coil conformation and high fluxionality. Antide fulfills the conformational requirements that are known to play a crucial role in receptor recognition, namely (i) the presence of a turn in the backbone and (ii) the all-trans nature of peptide bonds. In addition, the structural rigidity of antide likely adds a further contribution to the receptor binding affinity.

Helix-stabilizing nonpolar interactions between tyrosine and leucine in aqueous and TFE solutions: 2D-1H NMR and CD studies in alanine-lysine peptides.

Interactions between side chains spaced (i,i + 3) and (i,i + 4) may explain the context dependence of helix propensities observed in different systems. Nonpolar residues with these spacings occur frequently in protein helices and stabilize isolated peptide helices. Here (i,i + 3) and (i,i + 4) nonpolar interactions between Tyr and Leu in different solution conditions are studied in detail in alanine-based peptides using 2D 1H NMR and CD spectroscopy. Helix contents analyzed using current models for helix-coil transitions yield interaction energies which demonstrate significant helix stabilization in aqueous 1 M NaCl solutions by Tyr-Leu or Leu-Tyr pairs when spaced (i,i + 4) and, to a smaller extent, when spaced (i,i + 3), comparable to those estimated for other residue pairs. The interactions persist in solutions containing TFE, a helix-stabilizing solvent believed to diminish hydrophobic interactions, but not in helix-destabilizing 6 M urea. 1H NMR resonances for all peptides and solution conditions except in 6 M urea were completely assigned. NMR data indicate that the N-terminal residues are more helical and that the N-acetyl group participates in helix formation. The two (i,i + 4) spaced pairs show the same pattern of NOE cross-peaks between the Tyr and Leu side chains, as do the two (i,i + 3) pairs in 1 M NaCl as well in TFE solutions, and correspond well with that expected for the specific Tyr-Leu pair with side-chain contacts in protein helices.

Site-specific phosphorylation of Lys-Ser-Pro repeat peptides from neurofilament H by cyclin-dependent kinase 5: structural basis for substrate recognition.

Recent work has shown that high molecular weight neurofilament (NF) proteins are phosphorylated in their carboxy-terminal tail portion by the enzyme cyclin-dependent kinase 5 (CDK-5). The tail domain of neurofilaments contains 52 tripeptide repeats, viz. Lys-Ser-Pro, which mainly exist as KSPXK and KSPXXX motifs (X = amino acid). CDK-5 specifically phosphorylates the serine residues within the KSPXK sites. We probed the structural basis for this type of substrate selectivity by studying the conformation of synthetic peptides containing either KSPXK or KSPXXX repeats designed from native neurofilament sequences. Synthetic peptides with KSPXK repeats were phosphorylated on serine with a recombinant CDK-5/p25 complex whereas those with KSPXXX repeats were unreactive in this system. Circular dichroism (CD) studies in 50% TFE/H2O revealed a predominantly helical conformation for the KSPXXX-containing peptides, whereas the CD spectra for KSPXK-containing peptides indicated the presence of a high population of extended structures in water and 50% TFE solutions. However, detailed NMR analysis of one such peptide which included two such KSPXK repeats suggested a turn-like conformation encompassing the first KSPXK repeat. Restrained molecular dynamics calculations yielded an unusually stable, folded structure with a double "S"-like bend incorporating the central residues of the peptide. The data suggest that a transient reverse turn or loop-type structure may be a requirement for CDK-5-promoted phosphate transfer to neurofilament-specific peptide segments.

Mechanism of helix induction by trifluoroethanol: a framework for extrapolating the helix-forming properties of peptides from trifluoroethanol/water mixtures back to water.

To establish a framework for extrapolating the helix-forming properties of peptides from TFE/H2O mixtures (TFE = 2,2, 2-trifluoroethanol) back to water, the thermal unfolding curves have been measured by circular dichroism for four repeating-sequence peptides, with chain lengths from 7 to 22 residues. The unfolding curves were measured between 0 and 50 volume percent TFE and were fitted to the modified Lifson-Roig theory. A single set of helix-coil parameters fits the results for the four peptides at each TFE concentration; only two of the basic helix-coil parameters, <w>, the mean helix propagation parameter of residues in the sequence repeat, and DeltaH, the enthalpy change per residue on unfolding the helix, are allowed to vary with TFE molarity. The success in fitting these curves over a wide range of experimental variables shows that helix formation is basically the same in TFE/H2O mixtures as in water. Moreover, a simple model based on a linear dependence of ln <w> and DeltaH on TFE molarity can be used to extrapolate the results from 25% TFE (approximately 4 M) back to water. The results also give curves of helix formation induced by TFE at constant temperature, and the properties of these helix induction curves explain some of the puzzling results shown by other peptides in the literature. The average helix propensity increases regularly from 0 to 25% TFE but levels off at higher TFE concentrations, which explains why the extent of helix formation levels off in this range. The change in the apparent cooperativity of thermal unfolding curves in concentrated TFE solutions results from the decrease of the enthalpy change for helix unfolding at higher TFE concentrations. The rapid decrease in the plateau values of apparent helix content with increasing temperature results mainly from the strong temperature dependence of the ellipticity of the complete helix. To determine whether the helix-stabilizing effect of TFE arises from strengthening the hydrogen bonds in the helix backbone, the strength of the hydrogen bond in a model compound, salicylic acid, has been measured in TFE/H2O mixtures from the pKa difference between salicylic acid and a similar compound which cannot form the hydrogen bond. The curve of hydrogen bond strength versus increasing TFE concentration matches both in shape and magnitude the increase in average helix propensity in TFE/H2O mixtures.

An O-phosphotyrosine-containing analogue of human angiotensin ii exhibits increased serum stability

Abstract: Synthetic human [Tyr (P)]4-angiotensin II was analyzed by CD spectroscopy and compared with native human angiotensin II. The data showed there to be no apparent conformational difference between the two peptides in both aqueous and graded TFE solutions. An assessment of the in vitro stability of the two peptides to serum proteases showed clearly that the O-phosphorylated angiotensin II was more resistant to enzymic degradation suggesting that the tyrosyl O-phosphate plays a significant role in inhibiting this process.