Addition Of TFE Research Articles

Exploring the effect of hydrogen bonding on protonation of 7,8-benzoquinoline with TFE: Water binary mixture

In this work the role of hydrogen bonding in the protonation of 7,8-Benzoquinoline has been investigated using 2,2,2,-trifluoroethanol (TFE): water binary mixture. In our earlier work, protonation of 7,8-BQ was explored in water, TFE and acidic media. It appeared that protonation is significantly promoted in strong hydrogen bonding TFE. However, to substantiate it, a systematic study consisting of TFE: water binary was considered necessary. In this work, we observe that on addition of TFE in water, the intensity of neutral emission gradually decreases while the intensity of emission corresponding to protonated species increases due to faster and enhanced protonation as compared to pure water. Excited state properties of 7,8-BQ upon addition of TFE in water have been discussed using steady state and time resolved fluorescence spectroscopy. Quantum yield and excited state protonation rates have been calculated and discussed. We would like to mention that the present study validates our claim that hydrogen bonding indeed promotes protonation in 7,8-BQ. Besides the importance and applications of excited state proton transfer process in chemistry and biology the work has prospects in studying the conformational changes in proteins.

Probing interactions within liquid media via a model H-bond donor-acceptor mixture.

Understanding interactions within liquids so as to obtain desired physicochemical properties is of utmost importance in chemistry. Solute-solvent and solvent-solvent interactions within mixtures of hexamethylphosphoramide (HMPA) and 2,2,2-trifluoroethanol (TFE) are assessed using various invasive and non-invasive techniques. HMPA and TFE possess similar static dielectric constants. While HMPA has high H-bond accepting basicity (β ∼ 1) and no H-bond donating acidity (α ∼ 0), TFE has high H-bond donating acidity (α ∼ 1) and almost no H-bond accepting basicity (β ∼ 0). Kamlet-Taft parameters obtained using solvatochromic UV-Vis molecular absorbance probes suggest α and β of the (HMPA + TFE) mixture to be higher than those expected from mole-fraction weighted ideal-additive values with α and β exhibiting synergism in TFE-rich and HMPA-rich regions where α > 1 and β > 1, respectively. HMPA has lower dipolarity/polarizability (π*) than TFE, however, the addition of TFE to HMPA results in even further lowering of π* implying significant non-polar nature of the (HMPA + TFE) mixture as compared to its neat constituents. Photoinduced charge-transfer (neutral and anionic) and ratiometric solvatochromic fluorescence polarity probes also confirm the mixture dipolarity to be significantly lower than that expected. FTIR absorbance and Raman spectroscopic measurements hint at the weakening of the strong H-bonds among TFE and/or HMPA as the two solvents are mixed. Positive excess molar volumes (VE) and excess logarithmic viscosities [(ln η)E] indicate weakened interactions in the (HMPA + TFE) mixtures as compared to the interactions within neat TFE and neat HMPA. Microfluidity of the mixture obtained from an intramolecular excimer-forming fluorescence probe further supports these outcomes. Formation of a low polarity liquid milieu upon mixing of two higher polarity solvents is amply highlighted.

Retro operation on the Trp-cage miniprotein sequence produces an unstructured molecule capable of folding similar to the original only upon 2,2,2-trifluoroethanol addition.

Amino acid sequence and environment are the most important factors determining the structure, stability and dynamics of proteins. To evaluate their roles in the process of folding, we studied a retroversion of the well-described Trp-cage miniprotein in water and 2,2,2-trifluoroethanol (TFE) solution. We show, by circular dichroism spectroscopy and nuclear magnetic resonance (NMR) measurement, that the molecule has no stable structure under conditions in which the Trp-cage is folded. A detectable stable structure of the retro Trp-cage, with the architecture similar to that of the original Trp-cage, is established only upon addition of TFE to 30% of the total solvent volume. The retro Trp-cage structure shows a completely different pattern of stabilizing contacts between amino acid residues, involving the guanidinium group of arginine and the aromatic group of tryptophan. The commonly used online prediction methods for protein and peptide structures Robetta and PEP-FOLD failed to predict that the retro Trp-cage is unstructured under default prediction conditions. On the other hand, both methods provided structures with a fold similar to those of the experimentally determined NMR structure in water/TFE but with different contacts between amino acids.

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 Right-Side-Out Orientation of MscS in Liposomal Membranes

The bacterial mechanosensitive channel MscS plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. MscS functional characteristics have extensively been studied in both giant spheroplasts and liposomes. Despite many studies of MscS reconstituted into liposomes the channel orientation in liposomal membranes is still unknown. We examined the orientation of MscS in liposomes by patch-clamp and confocal microscopy. using its previously determined electrophysiological and pharmacological properties we were able to determine that in liposomes MscS retains the right-side-out orientation as in giant spheroplasts based on the following evidence: (i) I-V curves recorded in both spheroplast and liposome preparations exhibited strong outward rectification at both negative and positive pipette pressures. (ii) MscS activation ratio in liposome patches at positive relative to negative pipette voltages and vice versa showed positive correlation at both positive and negative pipette pressures similar to MscS in inside-out excised spheroplast patches. (iii) MscS exhibited a voltage-dependent hysteresis upon application of saw-tooth pressure ramps in both spheroplasts and liposomes. In both spheroplasts and liposomes the hysteresis was more pronounced upon positive pipette voltages compared to negative voltages. (iv) 2.5% of 2,2,2-trifluoroethanol (TFE) caused MscS inactivation in liposome patches when added to the cytoplasmic side of MscS, whereas addition of TFE to the periplasmic side did not inactivate the channel, although it caused a shift of the channel activation towards lower pipette pressures. We obtained a similar result when applying TFE to MscS in spheroplast patches. In conclusion, our findings strongly indicate that the cytoplasmic domain of MscS in liposome membrane patches faces the bath solution as in spheroplast patches. Consequently, upon liposome reconstitution MscS channels preserve their right-side-out orientation comparable to what was previously reported for the MscL channels.Supported by the NH & MRC.

Structural and Dynamics Characteristics of Acylphosphatase from Sulfolobus solfataricus in the Monomeric State and in the Initial Native-like Aggregates

It has previously been shown that the acylphosphatase from Sulfolobus solfataricus is capable of forming amyloid-like aggregates under conditions in which the native structure is maintained and via the transient formation of native-like aggregates. Based on the previously determined NMR structure of the native protein, showing a ferredoxin-like fold and the peculiar presence of an unstructured N-terminal segment, we show here, at a molecular level using NMR spectroscopy, that indeed S. solfataricus acylphosphatase remains in a native-like conformation when placed in aggregating conditions and that such a native-like structure persists when the protein forms the initial aggregates, at least within the low molecular weight species. The analysis carried out under different solution conditions, based on the measurement of the combined (1)H and (15)N chemical shifts and hydrogen/deuterium exchange rates, enabled the most significant conformational changes to be monitored upon transfer of the monomeric state into aggregating conditions and upon formation of the initial native-like aggregates. Important increases of the hydrogen/deuterium exchange rates throughout the native protein, accompanied by small and localized structural changes, in the monomeric protein were observed. The results also allow the identification of the intermolecular interaction regions within the native-like aggregates, that involve, in particular, the N-terminal unstructured segment, the apical region including strands S4 and S5 with the connecting loop, and the opposite active site.

Structure and dynamics changes induced by 2,2,2-trifluoro-ethanol (TFE) on the N-terminal half of hepatitis C virus core proteinThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

The Core protein of hepatitis C virus is involved in several interactions other than the encapsidation of viral RNA. We recently proposed that this is related to the fact that the N-terminal half of this protein (C82) is an intrinsically unstructured protein (IUP) domain. IUP domains can adopt a secondary structure when they are interacting with another molecule, such as a nucleic acid or a protein. It is also possible to mimic these conditions by modifying the environment of the protein. We investigated the propensity of this protein to fold as a function of salt concentration, detergent, pH, and 2,2,2-trifluoro-ethanol (TFE); only the addition of TFE resulted in a structural change. The effect of TFE addition was studied by circular dichroism, structural, and dynamic data obtained by NMR. The data indicate that C82 can adopt an alpha-helical structure; this conformation is likely relevant to one of the functional roles of the HCV Core protein.

Human tropoelastin sequence: Dynamics of polypeptide coded by exon 6 in solution

Calorimetric studies were performed on exon 6 in powdered form and in solution [water and 2,2,2-trifluoroethanol (TFE), a structure-inducing solvent or cosolvent]. Dynamic dielectric spectroscopy (DDS) analyses were realized in water and 20% TFE. The major role of solvent-peptide organization is evidenced with these techniques. Calorimetric measurements reveal the structural water organization around the polypeptide as well as the presence of hydrophobic interactions in TFE solution. Dielectric measurements showed for exon 6/water a decrease of relaxations times of bulk solvent implying a faster dynamics with a slight increase of the activation entropy, suggesting that exon 6 probably creates disorder within the solvent. For TFE/water mixtures, an influence of exon 6 on its environment was seen with a relaxation associated with the exon 6/solvent interactions reinforced by storage of 72 h. Finally, exon 6/solvent interactions were clearly observed with addition of TFE.

Electrospun Gelatin Fibers: Effect of Solvent System on Morphology and Fiber Diameters

Gelatin, a naturally-occurring biopolymer, was electrospun in the present contribution. Gelatin solutions were prepared in either single solvent system [i.e., glacial acetic acid (AA)] or mixed solvent systems [i.e., AA/2,2,2-trifluoroethanol (TFE), AA/dimethyl sulfoxide (DMSO), AA/ethylene glycol (EG), and AA/formamide (F)]. The electrospinning was carried out under a fixed electrostatic field strength of 7.5 kV/7.5 cm and the polarity of the emitting electrode was positive. The effects of these solvent systems on morphology and/or size of the electrospun materials were observed by scanning electron microscopy (SEM). Electrospinning of 15–29% w/v gelatin solutions in AA produced beads, beaded fibers, and smooth fibers, depending on the concentration range. Only smooth fibers were observed at the concentration range of 21–29% w/v, with their average diameter ranging between 214 and 839 nm. The addition of TFE as a co-solvent or another modifying liquid of DMSO, EG, or F helped improve the electrospinnability of the resulting gelatin solution. Among the three modifying liquids, DMSO and EG contributed to the formation of smooth gelatin fibers with reduced diameters when compared with those obtained from the solution in pure AA.

Structural Determinants That Target the Hepatitis C Virus Core Protein to Lipid Droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.

Dermaseptin S9, an α-Helical Antimicrobial Peptide with a Hydrophobic Core and Cationic Termini

The dermaseptins S are closely related peptides with broad-spectrum antibacterial activity that are produced by the skin of the South American hylid frog, Phyllomedusa sauvagei. These peptides are polycationic (Lys-rich), alpha-helical, and amphipathic, with their polar/charged and apolar amino acids on opposing faces along the long axis of the helix cylinder. The amphipathic alpha-helical structure is believed to enable the peptides to interact with membrane bilayers, leading to permeation and disruption of the target cell. We have identified new members of the dermaseptin S family that do not resemble any of the naturally occurring antimicrobial peptides characterized to date. One of these peptides, designated dermaseptin S9, GLRSKIWLWVLLMIWQESNKFKKM, has a tripartite structure that includes a hydrophobic core sequence encompassing residues 6-15 (mean hydrophobicity, +4.40, determined by the Liu-Deber scale) flanked at both termini by cationic and polar residues. This structure is reminiscent of that of synthetic peptides originally designed as transmembrane mimetic models and that spontaneously become inserted into membranes [Liu, L., and Deber, C. M. (1998) Biopolymers 47, 41-62]. Dermaseptin S9 is a potent antibacterial, acting on gram-positive and gram-negative bacteria. The structure of dermaseptin S9 in aqueous solution and in TFE/water mixtures was analyzed by circular dichroism and two-dimensional NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin S9 is aggregated in water, but a monomeric nonamphipathic alpha-helical conformation, mostly in residues 6-21, is stabilized by the addition of TFE. These results, combined with membrane permeabilization assays and surface plasmon resonance analysis of the peptide binding to zwitterionic and anionic phospholipid bilayers, demonstrate that spatial segregation of hydrophobic and hydrophilic/charged residues on opposing faces along the long axis of a helix is not essential for the antimicrobial activity of cationic alpha-helical peptides.

Remarkable Enhancement Effect of 2,2,2-Trifluoroethanol on the Solubility of Tris(pentane-2,4-dionato)chromium(III) in Supercritical Carbon Dioxide by Outer-Sphere Complexation through Hydrogen Bonding

Abstract The solubility of tris(pentane-2,4-dionato)chromium(III) (Cr(acac)3) in supercritical carbon dioxide (SC-CO2) with and without modifiers was investigated by UV–vis spectrophotometry. The influence of some polar and nonpolar modifiers, such as methanol, ethanol, 2,2,2-trifluoroethanol (TFE), acetone, chloroform, and benzene, was studied by changing the temperature, pressure, and concentration of the modifiers. Alcohols, particularly TFE, demonstrated a large solubility enhancement of Cr(acac)3 among these modifiers. In contrast, acetone having a relatively larger polarity gave a lower enhancement effect than chloroform and benzene. The measurement of the IR absorption spectra of TFE in the presence of Cr(acac)3 in SC-CO2 suggested that a drastic solubility enhancement of Cr(acac)3 upon the addition of TFE could be ascribed to the formation of the stable Cr(acac)3–TFE complex through hydrogen bonding. The association constants of Cr(acac)3 with TFE in SC-CO2 could be determined from the relationship of the solubility enhancement against the TFE concentration.

Helical structure of dermaseptin B2 in a membrane-mimetic environment.

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.

Extreme stability of helices formed by water‐soluble poly‐N‐substituted glycines (polypeptoids) with α‐chiral side chains

Poly-N-substituted glycines or "peptoids" are protease-stable peptide mimics. Although the peptoid backbone is achiral and lacks hydrogen-bond donors, substitution with alpha-chiral side chains can drive the formation of stable helices that give rise to intense CD spectra. To systematically study the solution properties and stability of water-soluble peptoid helices with alpha-chiral side chains, we have synthesized and characterized an amphipathic, 36-residue N-substituted glycine oligomer. CD was used to investigate effects of concentration and solvent environment on this helical peptoid. We saw no significant dependence of helical structure on concentration. Intense, "alpha-helix-like" CD spectra were observed for the 36-mer in aqueous, 2,2,2-trifluorethanol (TFE), and methanol solution, proving a relative insensitivity of peptoid helical structure to solvent environment. While CD spectra taken in these different solvents were fundamentally similar in shape, we did observe some interesting differences in the intensities of particular CD bands in the various solvents. For example, the addition of TFE to an aqueous solvent increases the degree of peptoid helicity, as is observed for polypeptide alpha-helices. Moreover, the helical structure of peptoids appears to be virtually unaffected by heat, even in an aqueous buffer containing 8 M urea. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role of steric forces in their structural stabilization. The structured polypeptoids studied here may have potential as robust mimics of helical polypeptides of therapeutic interest.

Solution structure of a peptide model of a region important for the folding of alpha-lactalbumin provides evidence for the formation of nonnative structure in the denatured state.

Elucidating the properties of the denatured state of proteins under conditions relevant for their folding is a key factor in understanding the folding process. We show that a peptide corresponding to residues 111-120 of human alpha-lactalbumin has a pronounced propensity to adopt nonnative structure in aqueous solution. Two-dimensional NMR provides evidence for a structured, nonnative conformation in fast exchange with a random coil ensemble. A total of 78 Rotating Frame Overhauser Effects (ROEs) were used to calculate the conformation of the structured population. A nonnative cluster of hydrophobic residues involving the side chains of K114, W118, Ll119, and A120 is observed, which helps to stabilize a turn-like conformation in the vicinity of residues 115-118. The structure in 30% (vol/vol) TFE was also calculated. Interestingly, the addition of TFE did not simply amplify the population of the structured conformer observed in H2O, but instead induced a new conformation. The implications for the folding of the intact protein are discussed. We also discuss the implications of this study for the relevance of the use of mixed TFE/H2O solvent systems to study isolated peptides.

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane-embedded G protein-coupled receptor (PTH1 Rc) present on the surface of cells in target tissues such as bone and kidney. This binding occurs in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34) PTH/PTHrP hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1-27)-[Tyr(34)]bPTH(28-34)-NH(2) (hybrid I) and PTHrP(1-18)-[Nal(23), Tyr(34)]bPTH(19-34)-NH(2) (hybrid II). Hybrid I is as active as PTH(1-34)NH(2) and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two-dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil-helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of approximately 35 and approximately 30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor.

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrP) point-mutated hybrids.

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34)PTH/PTHrP segmental hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point-mutated PTH/PTHrP 1-34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V(21)) and glutamic acid (hybrid E(19)), respectively, taken from the PTH sequence. Hybrid V(21) exhibits both high receptor affinity and biological potency, while hybrid E(19) binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2, 2-trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil-helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is approximately 80%. In the presence of DPC micelles, the maximum helix content is approximately 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D-nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N- and C-termini, was observed in both hybrids. However, the biologically potent hybrid V(21) exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E(19) are located at position 11 and in the sequence 20-26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor.

Conformation of four peptides corresponding to the α-helical segments of human GM–CSF

The conformation of segments corresponding to the four α-helical stretches found in human granulocyte-macrophage colony-stimulating factor was studied in water solution in the presence of different amounts of 2,2,2-trifluoroethanol (TFE). The CD spectra reveal the onset of secondary structure upon addition of TFE. The final amount of helical conformation varies among the four peptides. In all cases, the conformational transition is complete before 50% TFE (v/v). 1H-NMR studies were conducted at this solvent composition, leading to the assignment of all the resonances and to the definition of the secondary structure for all four fragments. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci.3: 336–346 No. of Figures: 12. No. of Tables: 5. No. of

High helicity of peptide fragments corresponding to β-strand regions of β-lactoglobulin observed by 2D-NMR spectroscopy

Whereas protein fragments, when they are structured, adopt conformations similar to that found in the native state, the high helical propensity of beta-lactoglobulin, a predominantly beta-sheet protein, suggested that the fragments of beta-lactoglobulin can assume the non-native helical conformation. In order to assess this possibility, we synthesized four 17-18-residue peptides corresponding to three beta-strand regions and one helical region (as a control) of beta-lactoglobulin and examined their conformation. We observed residual helicities of up to 17% in water, by far-UV CD, for all four peptide fragments. The helices could be significantly stabilized by the addition of TFE, and the NMR analyses in a mixture of 50% water/TFE indicated that helical structures are formed in the central region whereas both termini are frayed. Thus, the very same residues that form strands in the native beta-lactoglobulin showed high helical preferences. These results stand out from the current general view that peptide fragments isolated from proteins either are unfolded or adopt native-like secondary structures. The implications of the results in the mechanism of protein folding and in designing proteins and peptides are significant.

High Helical Propensity of the Peptide Fragments Derived from β-Lactoglobulin, a Predominantly β-sheet Protein

It is generally assumed that peptide fragments derived from globular proteins are either unfolded, or adopt native-like secondary structures, in particular α-helix, which are similar to those occurring in the early stages of protein folding. Since the structured conformations of short peptides are unstable, 2,2,2-trifluoroethanol (TFE) is often used to stabilize them. To examine the folding process of β-proteins, we synthesized three fragments of β-lactoglobulin corresponding to two β-strands and one helix region, and examined their conformation by circular dichroism and nuclear magnetic resonance. These regions were chosen because, according to secondary structure prediction, all three should have high helix propensities. In aqueous solution, the three peptides had only a little ordered structure, but when TFE was added, they exhibited marked helical propensities, as also observed for the whole molecule of β-lactoglobulin. These results indicate that the intrinsic helical propensity of a peptide fragment elucidated by the addition of TFE is not necessarily related to the secondary structure in the native state. The results further suggest a case of non-hierarchical protein folding model, in which non-native structures may be involved in the early stage of folding.