The effect of the cosolvent trifluoroethanol on a tryptophan side chain orientation in the hydrophobic core of troponin C

The unique biophysical properties of tryptophan residues have been exploited for decades to monitor protein structure and dynamics using a variety of spectroscopic techniques, such as fluorescence and nuclear magnetic resonance (NMR). We recently designed a tryptophan mutant in the regulatory N‐domain of cardiac troponin C (F77W‐cNTnC) to study the domain orientation of troponin C in muscle fibers using solid‐state NMR. In our previous study, we determined the NMR structure of calcium‐saturated mutant F77W‐V82A‐cNTnC in the presence of 19% 2,2,2‐trifluoroethanol (TFE). TFE is a widely used cosolvent in the biophysical characterization of the solution structures of peptides and proteins. It is generally assumed that the structures are unchanged in the presence of cosolvents at relatively low concentrations, and this has been verified for TFE at the level of the overall secondary and tertiary structure for several calcium regulatory proteins. Here, we present the NMR solution structure of the calcium saturated F77W‐cNTnC in presence of its biological binding partner troponin I peptide (cTnI_144–163) and in the absence of TFE. We have also characterized a panel of six F77W‐cNTnC structures in the presence and absence TFE, cTnI_144–163, and the extra mutation V82A, and used ¹⁹F NMR to characterize the effect of TFE on the F77(5fW) analog. Our results show that although TFE did not perturb the overall protein structure, TFE did induce a change in the orientation of the indole ring of the buried tryptophan side chain from the anticipated position based upon homology with other proteins, highlighting the potential dangers of the use of cosolvents.     

Organic cosolvents and hen egg white lysozyme folding

Studies on the influence of organic cosolvents on lysozyme folding have been reported. As most of the researches are confined to a few specific molecules and focus on equilibrium states, less is known about the effect on folding dynamics. We have studied the influence of six soluble organic cosolvents on hen egg white lysozyme heat induced denaturation and refolding dynamics. It was found that trifluoroethanol (TFE) can change the folding pathway significantly. With the presence of TFE, the overshot phenomenon generally observed in lysozyme folding at 222 nm disappears. The common mechanism of how organic cosolvents influence folding is analyzed. The heat induced denaturation temperature was found to have a quantitative relationship with the slow phase rate constant during folding. We discuss this finding and hypothesize that it is due to the similar influence of organic cosolvent on the transition state of heat denaturation and refolding.     

Cooperative alpha-helix formation of beta-lactoglobulin and melittin induced by hexafluoroisopropanol.

Alcohols denature the native state of proteins, and also stabilize the alpha-helical conformation in unfolded proteins and peptides. Among various alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) are often used because of their high potential to induce such effects. However, the reason why TFE and HFIP are more effective than other alcohols is unknown. Using CD, we studied the effects of TFE and HFIP as well as reference alcohols, i.e., methanol, ethanol, and isopropanol, on the conformation of bovine beta-lactoglobulin and the bee venom melittin at pH 2. Upon addition of alcohols, beta-lactoglobulin exhibited a transformation from the native state, consisting of beta-sheets, to the alpha-helical state, whereas melittin folded from the unfolded state to the alpha-helical state. In both cases, the order of effectiveness of alcohols was shown to be: HFIP > TFE > isopropanol > ethanol > methanol. The alcohol-induced transitions were analyzed assuming a two-state mechanism to obtain the m value, a measure of the dependence of the free energy change on alcohol concentration. Comparison of the m values indicates that the high potential of TFE can be explained by the additive contribution of constituent groups, i.e., F atoms and alkyl group. On the other hand, the high potential of HFIP is more than that expected from the additive effects, suggesting that the cooperative formation of micelle-like clusters of HFIP is important.     

Helix-enhancing propensity of fluoro and alkyl alcohols: influence of pH, temperature and cosolvent concentration on the helical conformation of peptides.

We have analyzed the effects of trifluoroethanol (TFE) and three other alcohols(1-propanol, 2-propanol and hexafluoro-2-propanol) on S-peptide (residues 1-20) of ribonuclease A, an analog of S-peptide (QHM-->AAA, Sa-peptide) and TC-peptide (residues 295-316) of thermolysin to assess the helix-enhancing propensity of fluoro and alkyl alcohols under different environmental conditions of cosolvent concentration, pH and temperature by circular dichroism (CD). The dependence of cosolvent concentration on helix-induction showed a plateauing effect in all cases. 1-Propanol and 2-propanol were as effective as TFE in all the three peptides. Hexafluoro-2-propanol (HFIP) was a better helix enhancer in all cases however, the relative effectiveness varied with the peptide sequence. The alcohol transitions were analyzed assuming a two-state transition. The free energy decreased linearly in the cosolvent concentration range of 0-5 m for all the three peptides. The m-value (constant of proportionality) varied between peptides but was similar for any given peptide for TFE, 1-propanol or 2-propanol. The m-values of HFIP for all three peptides was much higher compared to other cosolvents. The isothermal cosolvent helix-induction curves for the three peptides exhibited similar features of shape and character for 1-propanol, 2-propanol and TFE. The additivity of cosolvent-induced helix formation was observed for different blends of alkyl and/or fluoro cosolvents. The pH-dependence of helix formation was observed in both TFE and 1-propanol solutions for S-peptide and TC-peptide, respectively, while in Sa-peptide, which was designed to perturb the pH-effect, helix formation was unaffected. The overall results provide some insight into the mechanism of cosolvent-mediated helix-enhancement in protein segments and are likely to facilitate optimization of conditions for cosolvent usage in chemistry and biology.     

Alteration of heme axial ligands in hemoglobin by organic solvents analyzed by CD, FTIR, and XANES techniques

The effects of mixed solvents on the ligand binding site in hemoglobin have been investigated though three spectroscopic techniques. Two classes of organic solvents (amides and alcohols) known to increase or decrease the hemoglobin affinity have been chosen for this study. The analysis of the iron CO stretching band shows that the ligand binding sites of alpha CO and beta CO subunits inside the alpha 2 beta 2 hemoglobin tetramer exhibit multiple conformations. From the circular dichroism and X-ray absorption near-edge structure data, it appears that no core deformation or heme reorientation occur with the affinity changes. The iron-ligand average bond angle is the sole parameter that depends on the external solvent. Since cosolvents seem to affect the dynamics rather than the hindrance of the heme cavity, we suggest that the protein affinity could be associated with a hierarchy of subtle dynamic states.     

Electrostatics dominate quadruplex stability

Stabilization of nucleic acid structures results from a balance of multiple interactions, including electrostatics, base stacking, hydrophobic interactions, hydrogen bonding, van der Waals forces, etc. Nucleic acid quadruplexes are unusual structures in that their formation is driven by specific binding of metal ions. This unique mode of metal binding, which is tightly coupled to oligonucleotide folding, can engender correspondingly unique solution behavior. In particular, we show that addition of many cosolvents, such as primary aliphatic alcohols, increases the thermal stability of quadruplexes, as determined by melting temperature, Tm, in direct contrast to the response of duplexes to the same admixture of solvents. Thermal stability is observed to increase as the dielectric constant of the composite solvent decreases. This behavior suggests a dominant role for electrostatics in quadruplex formation and stability. Additional studies done with other cosolvents and solutes suggest that, in some cases, other forces may come into play, including the possibility of direct interaction with the quadruplex structure. Nonetheless, many cosolvents and small molecules, such as ethanol, dimethylformamide, and betaine, stabilize the quadruplex conformation in sharp distinction to their destabilization of DNA duplexes.     

Oxygen affinity of bovine haemoglobin. Relevance of electrostatic and hydrophobic interactions

We have studied the effects of organic cosolvents (monohydric alcohols and formamide) on the oxygen equilibrium of bovine haemoglobin and have compared them with the effects of the same cosolvents on the oxygen equilibrium of human haemoglobin. Our results indicate: (1) that in agreement with previous suggestions, the lower affinity of bovine haemoglobin for oxygen is not due to an increased number of salt bridges stabilizing the T structure; (2) that, following T----R transition, more hydrophobic surface is exposed to the solvent by bovine than by human haemoglobin. We suggest, therefore, that a relevant role in keeping the oxygen affinity of bovine haemoglobin lower than that of human haemoglobin is played by the higher free energy needed to expose this more hydrophobic surface to the solvent. We stress, however, that our analysis does not enable us to say which particular amino acid residues are concerned in these effects.     

The effect of organic cosolvents on the oxygen affinity of fetal hemoglobin. Relevance of protein-solvent interactions to the functional properties.

We have studied the effects of organic cosolvents (monohydric alcohols and formamide) on the oxygen affinity of human fetal hemoglobin stripped of phosphates and have compared them with the effects of the same cosolvents on the oxygen affinity of human adult hemoglobin under the same experimental conditions. Our results confirm that, in fetal hemoglobin, the T in equilibrium R conformational equilibrium is more displaced toward the T conformation than in the adult form and indicate that increased electrostatic and hydrophobic protein-solvent interactions contribute to this effect. The data reported are discussed in terms of the known amino acid substitutions between the beta- and gamma-chains and an attempt is made to rationalize the results with a molecular mechanism based on the crystallographic structure of fetal deoxyhemoglobin.     

Differential effects of alcohols on conformational switchovers in alpha-helical and beta-sheet protein models

Organic solvents may induce non-native structures of proteins that mimic folding intermediates and/or conformations that occur in proximity to biological membranes. Here we systematically investigate the effects of simple (i.e., MeOH and EtOH) and fluorinated (i.e., trifluoroethanol, TFE) alcohols on the secondary structure and thermodynamic stability of two complementary model proteins using a combination of circular dichroism, fluorescence, and Fourier transform infrared (FTIR) detection methods. The selected proteins are alpha-helical Borrelia burgdorferi VlsE and beta-sheet human mitochondrial co-chaperonin protein 10 (cpn10). We find that switches between VlsE's native and non-native superhelical and beta-sheet structures readily occur (pH 7, 20 degrees C). The pathway depends on the alcohol: addition of MeOH induces a transition to a superhelical structure that is followed by conversion to beta-structure, whereas EtOH only unfolds the protein. TFE unfolds VlsE at low percentages but promotes the formation of a superhelical state upon further additions. For cpn10, both MeOH and TFE additions govern initial unfolding; however, further additions of MeOH result in the formation of a non-native beta-structure, whereas subsequent additions of TFE induce a superhelical structure. EtOH additions promptly unfold and precipitate cpn10. Both VlsE's and cpn10's non-native structures exhibit high stability toward chemical and thermal perturbations. This study demonstrates that in response to different alcohols, polypeptides can readily adopt both alpha- and beta-enriched conformations. The biological significance of these findings is discussed.     

Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state

A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment. However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn. The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising, alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant. We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity.     

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 A), 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 (ASTm1-142, Tm143-284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm143-284(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 Tm143-284(5OHW269) with ASTm1-142 to calculate the dissociation constant (Kd) 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.     

Main-chain Dynamics of a Partially Folded Protein:15N NMR Relaxation Measurements of Hen Egg White Lysozyme Denatured in Trifluoroethanol

¹⁵N NMR relaxation measurements have been used to study the dynamic behaviour of the main-chain of hen lysozyme in a partially folded state, formed in a 70% (v/v) trifluoroethanol (TFE)/30% water mixture at 37°C and pH 2. This state is characterised by helical secondary structure in the absence of extensive tertiary interactions. The NMR relaxation data were interpreted by mapping of spectral density functions and by derivation of segmental as well as global order parameters. The results imply that the dynamics of lysozyme in TFE can, at least for the great majority of residues, be adequately described by internal motions which are superimposed on an overall isotropic tumbling of the molecule. Although the dynamic behaviour shows substantial variations along the polypeptide chain, it correlates well with the conformational preferences identified in the TFE state by other NMR parameters. Segments of the polypeptide chain which are part of persistent helical structures are highly restricted in their motion (S²> 0.8, with effective internal correlation times τ_e< 200 ps) but are also found to experience conformational exchange on a millisecond timescale. Regions which are stabilised in less persistent helical structure possess greater flexibility (0.6 <S²< 0.8, 200 ps < τ_e< 1 ns) and those which lack defined conformational preferences are highly flexible (S²< 0.6, τ_e∼1 ns). The dynamic behaviour of the main-chain was found to be correlated with other local features of the polypeptide chain, including hydrophobicity and the position of the disulphide bridges. Despite the absence of extensive tertiary interactions, preferential stabilisation of native-like secondary structure by TFE results in a pattern of main-chain dynamics which is similar to that of the native state.     

Comparison of the effects of 2,2,2-trifluoroethanol on peptide and protein structure and function

The co-solvent 2,2,2-trifluoroethanol (TFE) has been often used to aid formation of secondary structure in solution peptides or alternately as a denaturant within protein folding studies. Hen egg white lysozyme (HEWL) and a synthetic model peptide defining HEWL helix-4 were used as comparative model systems to systematically investigate the effect of increasing TFE concentrations on the structure of proteins and peptides. HEWL was analyzed using NMR, far-UV CD and fluorescence spectroscopy; with correlation of these results towards changes in enzymatic activity and the helix-4 peptide was analysed using NMR. Data illustrates two conflicting modes of interaction: Low TFE concentrations stabilize tertiary structure, observed from an increase in the number of NMR NOE contacts. Higher TFE concentrations denatured HEWL with the loss of lysozyme tertiary structure. The effects of TFE upon secondary structural elements within HEWL are distinct from those observed for the helix-4 peptide. This illustrates a dissimilar interaction of TFE towards both protein and peptide at equivalent TFE concentrations. The concentration that TFE promotes stabilization over denaturation is likely to be protein dependent although the structural action can be extrapolated to other protein systems with implications for the use of TFE in structural stability studies.     

Folding determinants of disulfide bond forming protein B explored by solution nuclear magnetic resonance spectroscopy

The two-stage model for membrane protein folding postulates that individual helices form first and are subsequently packed against each other. To probe the two-stage model, the structures of peptides representing individual transmembrane helices of the disulfide bond forming protein B have been studied in trifluoroethanol solution as well as in detergent micelles using nuclear magnetic resonance (NMR) and circular dichroism spectroscopy. In TFE solution, peptides showed well-defined α-helical structures. Peptide structures in TFE were compared to the structures of full-length protein obtained by X-ray crystallography and NMR. The extent of α-helical secondary structure coincided well, lending support for the two-stage model for membrane protein folding. However, the conformation of some amino acid side chains differs between the structures of peptide and full-length protein. In micellar solution, the peptides also adopted a helical structure, albeit of reduced definition. Using measurements of paramagnetic relaxation enhancement, peptides were confirmed to be embedded in micelles. These observations may indicate that in the native protein, tertiary interactions additionally stabilize the secondary structure of the individual transmembrane helices.     

Local structures in unfolded lysozyme and correlation with secondary structures in the native conformation: helix-forming or -breaking propensity of peptide segments

CD spectra of reduced and S-3-(trimethylated amino) propylated lysozyme (TMAP lysozyme) have been measured in various solutions containing guanidine hydrochloride or trifluoroethanol (TFE). The CD spectra indicate that there remain residual secondary structures in protein in aqueous solution. The addition of TFE further promotes the formation of secondary structures. In order to examine whether secondary structures are evenly induced over all the polypeptide chain, or locally at particular segments, the limited proteolysis of TMAP lysozyme by trypsin has been performed, and the CD spectra of all the final and intermediate products have been observed in solutions containing TFE. As a result, the fragments vary in a helix-forming propensity. The CD spectra of peptide fragments T5, T7, T9T10, T12T13, T14T15T16, and T17T18 are not significantly affected by the addition of TFE, where T refers to the nomenclature of R.E. Canfield [(1963), Journal of Biological Chemistry, Vol. 238, pp. 2691-2697]. They are fragments of a helix-breaking propensity. On the other hand, fragment I2 composed of T1-T4, and fragments T6T7, T8, and T11, attain secondary structures with the addition of TFE. They are fragments of a helix-forming propensity. Further, it is found that the fragments of a helix-forming propensity just correspond to the helical segments in native lysozyme. We examine the interactions between neighboring fragments, which contribute to the stabilization of local structures along the polypeptide chain.     

Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains

Several models for interactions between trifluoroethanol (TFE) and peptides and proteins have recently been proposed, but none have been able to rationalize the puzzling observations that on the one hand TFE can stabilize some hydrophobic interactions in secondary structures, but on the other can also melt the hydrophobic cores of globular proteins. The former is illustrated in this paper by the effect of TFE on a short elastin peptide, GVG(VPGVG)(3), which forms type II beta-turns stabilized by hydrophobic interactions between two intra-turn valine side chains. This folding, driven by increasing the entropy of bulk water, is stimulated in TFE-water mixtures and/or by raising the temperature. To explain these apparently contradictory observations, we propose a model in which TFE clusters locally assist the folding of secondary structures by first breaking down interfacial water molecules on the peptide and then providing a solvent matrix for further side chain--side chain interactions. This model also provides an explanation for TFE-induced transitions between secondary structures, in which the TFE clusters may redirect non-local to local interactions.     

Nonspecific interactions in dye binding to DNA. Influence of alcohols and amides

The binding of a few drugs (ethidium bromide, propidium diiodide, proflavine and actinomycin D) to DNA has been investigated in aqueous solutions to which cosolvents of different polarity have been added. It is found that both alcohols (less polar than water) and amides (more polar) lower the binding constant according to a linear relationship between the intercalation free energy and cosolvent concentration. The main action of cosolvents cannot be described in terms of electrostatic effects, since they predict much smaller changes in the binding constant than those observed. It appears instead that relevant solvation effects are responsible for the binding strength of the different dyes to DNA. As a general result, it is found that solvation effects largely contribute to the intercalation free energy, thereby weakening the influence of nonspecific interactions at the intercalation site.     

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. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 175–185, 2015.     

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.