Effect Of Trifluoroethanol Research Articles

Effect of trifluoroethanol on antibodies binding properties

The studies on the influence of organic co-solvents on the structure and function of antibodies are of key interest, especially in view of antibodies broad use as recognizing elements in different analytical systems. Here we studied the effect of co-solvent 2,2,2-trifluoroethanol (TFE) on the ability of anti-ovalbumin monoclonal antibodies to interact with its specific antigen. Antibody affinity to antigen and the rate constants of antibody binding to immobilized antigen were analyzed. Changes in antibody reactivity with incubation time which depended on TFE concentration and temperature were revealed. When treatment of antibodies with TFE was carried out at 0°C, we observed nonlinear, non-monotonous changes of antibody reactivity with initial fast decrease and substantial increase that may be related to the loss of antigen binding reactivity by some part of antibodies at the start but its restoration when the incubation proceeds. Keywords: 2;2;2-trifluoroethanol, antibody affinity, antigen-antibody interaction, monoclonal antibodies, ovalbumin

Effect of trifluoroethanol on antibody reactivity against corresponding and nonrelated antigens

Effect of trifluoroethanol on antibody reactivity against corresponding and nonrelated antigens

The Effects of Trifluoroethanol on the Helical Structure of SIKE

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure of SIKE. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To examine the structure of SIKE we have performed two dimensional proton NMR experiments and Circular Dichroism spectroscopy and compared the results with both secondary and tertiary structure predictions. 2D NOESY spectra obtained using several mixing times in general revealed little stable structure in the molecule as detected in the NH‐NH cross peak region, suggesting the protein may contain significant disordered regions. CD spectra suggest significant alpha helix content and the possibility of coiled coil regions of structure. Addition of Trifluoroethanol, known to stabilize alpha helical structures, led to increased CD signal at 222nm. Using a range of TFE concentrations optimal stabilization of helical structure in SIKE allowed comparison with helical secondary structure predications.Support or Funding InformationThis work was supported by NIH grant R21A1 107447 to JKB

Effect of trifluoroethanol on α-crystallin: folding, aggregation, amyloid, and cytotoxicity analysis.

α-Crystallin, a member of small heat shock proteins, is the major structural protein within the eye lens and is believed to play an exceptional role in the stability of lens proteins and its transparency. In the current manuscript, we have investigated the effect of an organic solvent, trifluoroethanol (TFE), on the structure and function of α-crystallin isolated from camel eye lens. Incubation of this protein with TFE changed the secondary and tertiary structures, which resulted in the aggregation of α-crystallin as evidenced by intrinsic fluorescence, Rayleigh's scattering, Thioflavin T assay, and circular dichroism spectroscopic studies. The treatment with different concentrations of TFE led to increased exposure of hydrophobic domains of α-crystallin, which was observed by 8-anilino 1-napthalene sulfonic acid extrinsic fluorescence assay. These results clearly indicate that TFE induced significant changes in the secondary and tertiary structures of α-crystallin, leading to aggregation and amyloid formation. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay established the cytotoxicity of the aggregated α-crystallin towards HepG2 cell lines through reactive oxygen species production. In conclusion, α-crystallin protein was found to be susceptible to conformational changes by TFE, suggesting that α-crystallin, although basically acting like a heat shock protein and functionally displaying chaperone-like activity, might capitulate to change in lens environment induced by diseased conditions or age-related changes, resulting in cataract formation.

Beaded nanofibers assembled from double-hydrophobic elastin-like block polypeptides: Effects of trifluoroethanol.

A "double-hydrophobic" elastin-like triblock polypeptide GPG has been constructed by mimicking the localization of proline- and glycine-rich hydrophobic domains of native elastin, a protein that provides elasticity and resilience to connective tissues. In this study, the effects of trifluoroethanol (TFE), an organic solvent that strongly affects secondary structures of polypeptides on self-assembly of GPG in aqueous solutions were systematically studied. Beaded nanofiber formation of GPG, where nanoparticles are initially formed by coacervation of the polypeptides followed by their connection into one-dimensional nanostructures, is accelerated by the addition of TFE at the concentrations up to 30% (v/v), whereas aggregates of nanoparticles are formed at 60% TFE. The concentration-dependent assembly pattern discussed is based on the influence of TFE on the secondary structures of GPG. Well-defined nanofibers whose diameter and secondary structures are controlled by TFE concentration may be ideal building blocks for constructing bioelastic materials in tissue engineering.

Isocyanurate based fluorinated polyurethane dispersion for anti-graffiti coatings

Fluorinated polyurethane dispersions (FPUDs) were synthesized using HDI isocyanurate, polyester polyol and DMPA with varying concentrations of trifluoro ethanol (TFE) in the range of 0–9wt%. Further FPUDs were characterized by FT-IR and 1H NMR for structural elucidation. Effects of trifluoro ethanol on the dispersion were evaluated by particle size analyzer. It was observed that with increase in TFE, particle size increases. Topography and contact angle were studied by atomic force microscopy (AFM) and goniometer, respectively. To gain more insight into the formation of roughness, power spectral density (PSD) analysis was carried out. It was found that with increase in TFE content, surface roughness increases due to migration of fluorinating agent on the coating substrate and consequently water repellency increases. Nano indentation of coating revealed that fluorinated coating possesses less stiffness and elastic modulus, due to the presence of fluorine on the coating. Further, coatings were analyzed for thermal properties by thermogravimetry analysis (TGA) and differential scanning calorimetry (DSC) which shows the enhancement in thermal stability and glass transition temperature with increase in TFE content. Anti-graffiti test was carried out which showed improved resistance with increasing fluorine content.

Trifluoroethanol and acetonitrile induced formation of the molten globule states and aggregates of cellulase

A systematic investigation on the effects of trifluoroethanol and acetonitrile at various concentrations on cellulase (EC 3.2.1.4) was studied by enzyme assay, intrinsic fluorescence, ANS binding, circular dichroism and ATR-Fourier transform infra red spectroscopy. The results show the presence of molten globule states at 3% (v/v) TFE and 80% (v/v) ACN. Cellulase aggregates at 25% (v/v) TFE and 90% (v/v) ACN, as detected by decrease in intrinsic and ANS fluorescence, loss in tertiary structure and enzyme activity, increase non-native β-sheet structure as evident from far-UV CD and FTIR spectra, increase in Thioflavin T fluorescence and shift in Congo red assay.

Trifluoroethanol-Induced Changes in Activity and Conformation of Manganese-Containing Superoxide Dismutase

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important role in antioxidant defense in organisms exposed to oxygen. However, there is a lack of research into the regulation of SOD activity and structural changes during folding, especially for SOD originating from extremophiles. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of manganese-containing SOD (Mn-SOD) from Thermus thermophilus. TFE decreased the degree of secondary structure of Mn-SOD, which directly resulted in enzyme inactivation and disrupted the tertiary structure of Mn-SOD. The kinetic studies showed that TFE-induced inactivation of Mn-SOD is a first-order reaction and that the regional Mn-contained active site is very stable compared to the overall structure. We further simulated the docking between Mn-SOD and TFE (binding energy for Dock 6.3, -9.68 kcal/mol) and predicted that the LEU9, TYR13, and HIS29 residues outside of the active site interact with TFE. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of TFE and allow us to describe ligand binding via inhibition kinetics combined with computational predictions.

Conformational changes during amyloid fibril formation of pancreatic thiol proteinase inhibitor: effect of copper and zinc

Pancreatic thiol proteinase inhibitor (PTPI), a variant of cystatin superfamily of cysteine protease inhibitors, has been isolated from pancreas of Capra hircus. In the present study, we examined the effects of acid denaturation and a co-solvent on PTPI with a focus on protein conformational changes and amyloid fibril formation. The results demonstrate that PTPI can form amyloid like fibrils. Acid denaturation as studied by CD and fluorescence spectroscopy showed that PTPI populates three partly unfolded species, a native like state at pH 3.0, a structured molten globule at pH 1.0 and partly unfolded species at pH 2.0, from each of which amyloid like fibrils grow as assessed by Thioflavin T (ThT) spectroscopy. Effect of trifluoroethanol (TFE) on acid induced states of PTPI was analyzed. TFE stabilized each of the three acid-induced intermediates at predenaturational concentrations (10%) and accelerated fibril formation. Morphology of the protein species at the beginning and end of reactions was observed using transmission electron microscopy. Solvent conditions were decisive for final fibril morphology. Biometals, Cu(2+) and Zn(2+) produced a concentration dependent decline in ThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM Cu(2+) or 10 μM Zn(2+) prevented any amyloid aggregation. Implications for therapeutics in view of Cu(2+) and Zn(2+) as essential micronutrients are suggested.

Effect of Trifluoroethanol on the Tryptophan Side Chain Orientation in the Hydrophobic Core of Troponin C Studied by NMR and Time Resolved Fluorescence

Several NMR studies have shown that co-solvent trifluoroethanol (TFE) does not perturb the overall three-dimensional structure of proteins. We have inserted a single tryptophan (F77W) in the hydrophobic core of the N-domain of cardiac troponin C (cNTnC), and determined the structure of the mutant with and without TFE (Julien et al., Protein Sci. 2009 18:1165-74). Interestingly, the position of the tryptophan side chain orientation was shown to be in opposite directions. We have monitored the effect of TFE on the tryptophan rotamer population using 13C-HSQC spectra and used a full lineshape analysis to quantify the rate of the conformational exchange.To further characterize this phenomenon, we have used Time Correlated Single Photon Counting experiments as a function of the TFE concentration. The time dependence could be fitted very well with three lifetimes in the wavelength region from 320 to 380 nm, and global analysis was further used. Addition of TFE (up to 19%) causes a gradual decrease of the lifetimes, due to dynamic quenching with very low quenching constants between kq = 0.1 to 0.01 M-1 ns-1. The amplitude fractions of the lifetimes change upon addition of TFE. At 340 nm, the amplitude fraction of the long lifetime (5.9 ns) increased from 13 to 29%, while that of the middle lifetime (3.3 ns) decreased from 63 to 50%. The short lifetime changed only to a limited extend. These data indicate that the change in the tryptophan indole position (different rotameric state) upon addition of TFE, as observed in NMR, is reflected mainly in the amplitude fractions of the different lifetimes. This is consistent, in this case, with the interpretation of lifetimes linked to rotameric states of tryptophan.

Adsorption and Diffusion of Plasma Proteins on Hydrophilic and Hydrophobic Surfaces: Effect of Trifluoroethanol on Protein Structure

The aim of this work was to investigate the conformational changes and diffusion of adsorbed proteins (immunoglobulin G (IgG), fibrinogen (Fib) and human serum albumin (HSA)) on hydrophilic quartz and hydrophobized quartz (octadecyltrichlorosilane (OTS)) surfaces. Circular dichroism spectroscopy measurements have shown that IgG is the most stable protein after adsorption on hydrophilic quartz, whereas HSA and Fib unfold. The structural changes are dependent on adsorption time, initial protein concentration in bulk, and surface chemistry. The effect of trifluoroethanol (TFE) in recovering the original protein structure after adsorption was analyzed by total internal reflection fluorescence and fluorescence recovery after photobleaching (TIRF-FRAP). TIRF-FRAP experiments revealed a strong dependence of the surface chemistry on protein diffusion coefficients: proteins diffuse 4 times slower on hydrophobic surfaces than on hydrophilic surfaces. The diffusion coefficient of TFE at hydrophobic surfaces is 2 orders magnitude higher than at hydrophilic surfaces. However, protein desorption occurs faster on hydrophilic quartz than on OTS, proving that the strength of protein-surface interaction is weaker at hydrophilic surfaces. This result shows that desorption is determined by surface/protein chemistry and not by mass transfer limitations. FTIR-ATR results demonstrated that TFE interaction with adsorbed proteins is stronger at hydrophilic surfaces than at hydrophobic surfaces.

A MOLECULAR DYNAMICS SIMULATION STUDY OF CONFORMATIONAL CHANGES AND SOLVATION OF Aβ PEPTIDE IN TRIFLUOROETHANOL AND WATER

Molecular dynamics (MD) simulations of amyloid beta peptide have been performed in aqueous solutions of trifluoroethanol with different concentrations. The amount of α-helical secondary structure increases when going from pure water to trifluoroethanol-rich solutions. The conformation obtained in 40% (v/v) trifluoroethanol solution is very similar to the experimental observations of beta peptide in sodium dodecyl sulfate micelle. In this solution, the peptide has two helical segments connected through a looped region. The C-terminal helix of beta peptide unfolds in pure water. The effect of trifluoroethanol on peptide's secondary structure has been explained using the properties calculated from MD trajectories.

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.

Different Effects of Trifluoroethanol and Glycerol on the Stability of Tropomyosin Helices and the Head-to-Tail Complex

Tropomyosin (Tm) is a dimeric coiled-coil protein, composed of 284 amino acids (410 Å), that forms linear homopolymers through head-to-tail interactions at low ionic strength. The head-to-tail complex involves the overlap of approximately nine N-terminal residues of one molecule with nine C-terminal residues of another Tm molecule. In this study, we investigate the influence of 2,2,2-trifluoroethanol (TFE) and glycerol on the stability of recombinant Tm fragments (ASTm 1–142, Tm 143–284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm 143–284(5OHW269)) contains a 5-hydroxytryptophan (5OHW) probe at position 269 whose fluorescence is sensitive to the head-to-tail interaction and allows us to accompany titrations of Tm 143–284(5OHW269) with ASTm 1–142 to calculate the dissociation constant ( K d) and the interaction energy at TFE and glycerol concentrations between 0% and 15%. We observe that TFE, but not glycerol, reduces the stability of the head-to-tail complex. Thermal denaturation experiments also showed that the head-to-tail complex increases the overall conformational stability of the Tm fragments. Urea and thermal denaturation assays demonstrated that both TFE and glycerol increase the stability of the isolated N- and C-terminal fragments; however, only TFE caused a significant reduction in the cooperativity of unfolding these fragments. Our results show that these two cosolvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their opposing effects on head-to-tail complex formation.

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of alpha-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.

Characterisation of Cytochrome c Unfolding by Nano-Electrospray Ionization Time-of-Flight and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Protein charge-state distributions (CSDs) in electrospray-ionization mass spectrometry (ESI-MS) represent a sensitive tool to probe different conformational states. We describe here the effect of trifluoroethanol (TFE) on cytochrome c equilibrium unfolding at different pH by nano-ESI-MS. While even low concentrations of TFE destabilize the protein native structure at low pH, a TFE content of 2.5%-5% is found to favor cyt c folding at pH approximately 7. Furthermore, we perform comparison of CSDs obtained by time-of-flight (ToF) and Fourier-transform-ion- cyclotron-resonance (FT-ICR) mass analyzers. To this purpose, we analyze spectra of cyt c in the presence of different kind of denaturants. In particular, experiments with 1-propanol suggest that also by FT-ICR-MS, as previously observed on an ESI-ToF instrument, CSDs do not appear to be controlled by the solvent surface tension as predicted by the Rayleigh-charge model. Moreover, there is general good agreement in conformational effects revealed by the different instruments under several buffer conditions. Nevertheless, the ToF instrument appears to discriminate better between unfolded and partially unfolded forms.

Glycosaminoglycans enhance the trifluoroethanol-induced extension of beta 2-microglobulin-related amyloid fibrils at a neutral pH.

beta(2)-Microglobulin-related (A beta 2M) amyloidosis is a frequent and serious complication in patients on long-term dialysis, and beta(2)-microglobulin is a major structural component of A beta 2M amyloid fibrils. Several biologic molecules inhibiting the depolymerization of A beta 2M amyloid fibrils at a neutral pH were found recently. The effect of trifluoroethanol and glycosaminoglycans (GAG) on the extension of the fibrils at a neutral pH was investigated with the use of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. Trifluoroethanol at concentrations of up to 20% (vol/vol) caused fibril extension of heparin-stabilized seeds, inducing a subtle change in the tertiary structure of beta(2)-microglobulin and stabilizing the fibrils at a neutral pH. This extension reaction followed a first-order kinetic model. In addition, some GAG, especially heparin, dose-dependently enhanced the fibril extension. These results suggest that some GAG, especially heparin, may bind to the fibrils and enhance their deposition in vivo. Thus, the experimental system described here should be useful to search for the factors that accelerate A beta 2M amyloid deposition in vivo. In addition, the interference of the binding of GAG to A beta 2M amyloid fibrils may be an attractive therapeutic modality.

Effect of trifluoroethanol on the conformational stability of a hyperthermophilic esterase: a CD study

The conformational stability of the hyperthermophilic esterase AFEST from Archeoglobus fulgidus against the denaturing action of 2,2,2-trifluoroethanol (TFE) has been investigated by means of circular dichroism (CD) measurements. At room temperature far-UV and near-UV CD spectra point out the occurrence of a co-operative transition from the native structure to a denatured state characterized by a high content of α-helix. The TFE concentration at half-completion of the transition proves to be 3.5 M (25% v v −1), by recording the molar ellipticity at both 222 and 276 nm. Thermal transition curves of AFEST in the absence and in the presence of TFE indicate a significant stability decrease on increasing the TFE concentration. The denaturation temperature is 99 °C for native AFEST, but becomes 85 °C at 1.4 M TFE (10% v v −1), and 56 °C at 2.8 M TFE (20% v v −1). It is also shown that, even though AFEST is very resistant to temperature, its resistance towards the denaturing action of TFE is similar to that of mesophilic proteins, including an esterase from Escherichia coli, AES. The proposal of a general mechanism for the TFE action on globular proteins leads to a reliable rationale of experimental data.

Synthesis and conformational characterization of peptides related to the neck domain of a fungal kinesin.

The Y362K mutation in the neck domain of conventional kinesin from Neurospora crassa provokes a significant reduction of the rate of movement along microtubules. Since the alpha-helical coiled-coil structure of the neck region is implicated in the mechanism of the processive movement of kinesins, a series of peptides related to the heptad region 338-379 of the wild-type and the variant fungal kinesinswere synthesized as monomers and as N-terminal disulfide dimers, crosslinked to favour self-association into coiled-coil structures entropically. A comparison of the dichroic properties of the peptides and the effects of trifluoroethanol and peptide concentration clearly confirmed the strong implication of the single point mutation in destabilizing the intrinsic propensity of the peptides to fold into the supercoiled conformation. That there is a correlation between the stability of the coiled-coil and rate of movement of the kinesin is confirmed.

Activity and conformational changes of horseradish peroxidase in trifluoroethanol.

The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5-25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25-40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The alpha-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.