Upgrading of the general AMBER force field 2 for fluorinated alcohol biosolvents: A validation for water solutions and melittin solvation.

The standard GAFF2 force field parameterization has been refined for the fluorinated alcohols 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and 1,1,1,3,3,3-hexafluoropropan-2-one (HFA), which are commonly used to study proteins and peptides in biomimetic media. The structural and dynamic properties of both proteins and peptides are significantly influenced by the biomimetic environment created by the presence of these cosolvents in aqueous solutions. Quantum mechanical calculations on stable conformers were used to parameterize the atomic charges. Different systems, such as pure liquids, aqueous solutions, and systems formed by melittin protein and cosolvent/water solutions, have been used to validate the new models. The calculated macroscopic and structural properties are in agreement with experimental findings, supporting the validity of the newly proposed models.

A three-state mechanism for trifluoroethanol denaturation of an intrinsically disordered protein (IDP).

Relating the amino acid composition and sequence to chain folding and binding preferences of intrinsically disordered proteins (IDPs) has emerged as a huge challenge. While globular proteins have respective 3D structures that are unique to their individual functions, IDPs violate this structure-function paradigm because rather than having a well-defined structure an ensemble of rapidly interconverting disordered structures characterize an IDP. This work measures 2,2,2-trifluoroethanol (TFE)-induced equilibrium transitions of an IDP called AtPP16-1 (Arabidopsis thaliana phloem protein type 16-1) by using fluorescence, circular dichroism, infrared and nuclear magnetic resonance (NMR) methods at pH 4, 298 K. Low TFE reversibly removes the tertiary structure to produce an ensemble of obligate intermediate ($\mathrm{I}$) retaining the native-state ($\mathrm{N}$) secondary structure. The intermediate $\mathrm{I}$ is preceded by a non-obligate tryptophan-specific intermediate ${\mathrm{I}}_{\mathrm{w}}$ whose population is detectable for AtPP16-1 specifically. Accumulation of such non-obligate intermediates is discriminated according to the sequence composition of the protein. In all cases, however, a tertiary structure-unfolded general obligate intermediate $\mathrm{I}$ is indispensable. The $\mathrm{I}$ ensemble has higher helical propensity conducive to the acquisition of an exceedingly large level of α-helices by a reversible denaturation transition of $\mathrm{I}$ to the denatured state $\mathrm{D}$ as the TFE level is increased. Strikingly, it is the same $\mathrm{N}\rightleftharpoons \mathrm{I}\rightleftharpoons \mathrm{D}$ scheme typifying the TFE transitions of globular proteins. The high-energy state $\mathrm{I}$ characterized by increased helical propensity is called a universal intermediate encountered in both genera of globular and disordered proteins. Neither $\mathrm{I}$ nor $\mathrm{D}$ strictly show molten globule (MG)-like properties, dismissing the belief that TFE promotes MGs.

A proof-of-concept study of the secondary structure of influenza A, B M2 and MERS- and SARS-CoV E transmembrane peptides using folding molecular dynamics simulations in a membrane mimetic solvent.

Here, we have carried out a proof-of-concept molecular dynamics (MD) simulation with adaptive tempering in a membrane mimetic environment to study the folding of single-pass membrane peptides. We tested the influenza A M2 viroporin, influenza B M2 viroporin, and protein E from coronaviruses MERS-Cov-2 and SARS-CoV-2 peptides with known experimental secondary structures in membrane bilayers. The two influenza-derived peptides are significantly different in the peptide sequence and secondary structure and more polar than the two coronavirus-derived peptides. Through a total of more than 50 µs of simulation time that could be accomplished in trifluoroethanol (TFE), as a membrane model, we characterized comparatively the folding behavior, helical stability, and helical propensity of these transmembrane peptides that match perfectly their experimental secondary structures, and we identified common motifs that reflect their quaternary organization and known (or not) biochemical function. We showed that BM2 is organized into two structurally distinct parts: a significantly more stable N-terminal half, and a fast-converting C-terminal half that continuously folds and unfolds between α-helical structures and non-canonical structures, which are mostly turns. In AM2, both the N-terminal half and C-terminal half are very flexible. In contrast, the two coronavirus-derived transmembrane peptides are much more stable and fast helix-formers when compared with the influenza ones. In particular, the SARS-derived peptide E appears to be the fastest and most stable helix-former of all the four viral peptides studied, with a helical structure that persists almost without disruption for the whole of its 10 µs simulation. By comparing the results with experimental observations, we benchmarked TFE in studying the conformation of membrane and hydrophobic peptides. This work provided accurate results suggesting a methodology to run long MD simulations and predict structural properties of biologically important membrane peptides.

Dependence of crystallographic atomic displacement parameters on temperature (25-150†K) for complexes of horse liver alcohol dehydrogenase.

Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1-1.3 Šresolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13-16 Å2) over the range 25-100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7-10 Å2 for the overall complexes and of 5-10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Šand is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.

Infrared spectroscopy and theoretical structure analyses of protonated fluoroalcohol clusters: the impact of fluorination on the hydrogen bond networks.

To explore the impact of fluorination on the hydrogen bond networks of protonated alkylalcohols, infrared spectroscopy and theoretical computations of protonated 2,2,2-trifluoroethanol clusters, H+(TFE)n, (n = 4-7), were performed. It has been demonstrated that the development of the hydrogen bond networks from a linear type to cyclic types occurs in this size region for the protonated alkylalcohol clusters. In contrast, infrared spectroscopy of H+(TFE)n in the OH/CH stretch region clearly indicated that the linear type structures are held in the whole size range, irrespective of temperature of the clusters. The extensive stable isomer structure search of H+(TFE)n based on our latest sampling approach supported the strong preference of the linear type hydrogen bond networks. Detailed analyses of the free OH stretching vibrational bands evidenced the intra- and intermolecular OH⋯FC interactions in the clusters. In addition, infrared spectra of protonated clusters of 2,2-difluoroethanol, 2,2-difluoropropanol, and 3,3,3-trifluoropropanol were measured for n = 4 and 5, and their spectra also indicated the effective inhibition of the cyclic hydrogen bond network formation by the fluorination.

Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca.

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defence peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-[Formula: see text] mixture. The structure is defined by an [Formula: see text]-helix that spans between Ile[Formula: see text]-Ala[Formula: see text], and a cyclic disulfide-bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide's interactions with sodium dodecyl sulfate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its [Formula: see text]-helical conformation between residues Ile[Formula: see text]-Ala[Formula: see text], while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

Correlating solvation with conformational pathways of proteins in alcohol-water mixtures: a THz spectroscopic insight.

Water, being an active participant in most of the biophysical processes, is important to trace how protein solvation changes as its conformation evolves in the presence of solutes or co-solvents. In this study, we investigate how the secondary structures of two diverse proteins - lysozyme and ß-lactoglobulin - change in the aqueous mixtures of two alcohols - ethanol and 2,2,2-trifluoroethanol (TFE) using circular dichroism measurements. We observe that these alcohols change the secondary structures of these proteins and the changes are protein-specific. Subsequently, we measure the collective solvation dynamics of these two proteins both in the absence and in the presence of alcohols by measuring the frequency-dependent absorption coefficient (α(ν)) in the THz (0.1-1.2 THz) frequency domain. The alcohol-water mixtures exhibit a non-ideal behaviour with the highest absorption difference (Δα) obtained at Xalcohol = 0.2. The protein solvation in the presence of the alcohols shows an oscillating behaviour in which Δαprotein changes with Xalcohol. Such an oscillatory behaviour of protein solvation results from a delicate interplay between the protein-water, protein-alcohol and water-alcohol associations. We attempt to correlate the various structural conformations of the proteins with the associated solvation.

Preparation and Synthetic Application of Naproxen-Containing Diaryliodonium Salts.

The synthesis of naproxen-containing diaryliodonium salts has been realized from naproxen methyl ester and ArI(OH)OTs activated by trimethylsilyl trifluoromethanesulfonate (TMSOTf) in a solvent mixture comprising dichloromethane and 2,2,2-trifluoroethanol (TFE). Those iodonium salts have been successfully used in the functionalization of an aromatic ring of naproxen methyl ester, including fluorination, iodination, alkynylation, arylation, thiophenolation, and amination and esterification reactions. Moreover, further hydrolysis of the obtained 5-iodo-naproxen methyl ester afforded 5-iodo-naproxen.

Changes in the secondary structures and zeta potential of soybean peptide and its calcium complexes in different solution environments.

To illustrate the relationship between environment hydrophobicity and soybean peptide and its calcium complexes when they are absorbed transmembrane, different solution environments (HBS buffer, TFE hydrophobic solution and cell suspension) were used to simulate hydrophilic and hydrophobic environments. In this study, soybean peptides (10-30 kDa) with a high calcium binding capacity were prepared by enzymatic hydrolysis and ultrafiltration. The results of cell experiments showed that the peptide could transport calcium into cells for absorption. Secondary structure changes of the peptide and its calcium complexes in different solution environments showed that the secondary structure of the peptide changed during the transmembrane absorption, and the contents of α-helix and ß-sheet structures increased. Besides, the ß-sheet structures in the peptide-calcium complexes were further converted to an α-helix structure. This conversion may be induced by the hydrophobicity of peptide solutions. In addition, when the conformation changes, the positively charged peptides in the sample will be exposed and then interact with cells, which is beneficial for the transmembrane of peptide-calcium complexes.

The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice.

Alpha-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and ß-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and ß-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.

Two different regimes in alcohol-induced coil-helix transition: effects of 2,2,2-trifluoroethanol on proteins being either independent of or enhanced by solvent structural fluctuations.

Inhomogeneous distribution of constituent molecules in a mixed solvent has been known to give remarkable effects on the solute, e.g., conformational changes of biomolecules in an alcohol-water mixture. We investigated the general effects of 2,2,2-trifluoroethanol (TFE) on proteins/peptides in a mixture of water and TFE using melittin as a model protein. Fluctuations and Kirkwood-Buff integrals (KBIs) in the TFE-H2O mixture, quantitative descriptions of inhomogeneity, were determined by small-angle X-ray scattering investigation and compared with those in the aqueous solutions of other alcohols. The concentration fluctuation for the mixtures ranks as methanol < ethanol ≪ TFE < tert-butanol < 1-propanol, indicating that the inhomogeneity of molecular distribution in the TFE-H2O mixture is unexpectedly comparable to those in the series of mono-ols. On the basis of the concentration dependence of KBIs between the TFE molecules, it was found that a strong attraction between the TFE molecules is not necessarily important to induce helix conformation, which is inconsistent with the previously proposed mechanism. To address this issue, by combining the KBIs and the helix contents reported by the experimental spectroscopic studies, we quantitatively evaluated the change in the preferential binding parameter of TFE to melittin attributed to the coil-helix transition. As a result, we found two different regimes on TFE-induced helix formation. In the dilute concentration region of TFE below ∼2 M, where the TFE molecules are not aggregated among themselves, the excess preferential binding of TFE to the helix occurs due to the direct interaction between them, namely independent of the solvent fluctuation. In the higher concentration region above ∼2 M, in addition to the former effect, the excess preferential binding is significantly enhanced by the solvent fluctuation. This scheme should be held as general cosolvent effects of TFE on proteins/peptides.

Molecular Diversity via Tetrasubstituted Alkenes Containing a Barbiturate Motif: Synthesis and Biological Activity.

The synthesis of a molecularly diverse library of tetrasubstituted alkenes containing a barbiturate motif is described. Base-induced condensation of N1-substituted pyrimidine-2,4,6(1H,3H,5H)-triones with 5-(bis(methylthio)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione gave 3-substituted 5-(methylthio)-2H-pyrano[2,3-d]pyrimidine-2,4,7(1H,3H)-triones ('pyranopyrimidinones'), regioselectively. A sequence of reactions involving ring-opening of the pyran moiety, displacement of the methylthio group with an amine, re-formation of the pyran ring, and after its final cleavage with an amine, gave tetrasubstituted alkenes (3-amino-3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)propanamides) with a diversity of substituents. Cleavage of the pyranopyrimidinones with an aniline was facilitated in 2,2,2-trifluoroethanol under microwave irradiation. Compounds were tested against Escherichia coli, Staphylococcus aureus, the yeast Schizosaccharomyces pombe, and the pathogenic fungus Candida albicans. No compounds exhibited activity against E. coli, whilst one compound was weakly active against S. aureus. Three compounds were strongly active against S. pombe, but none was active against C. albicans.

Synthesis of α-Aminophosphonic Acid Derivatives Through the Addition of O- and S-Nucleophiles to 2H-Azirines and Their Antiproliferative Effect on A549 Human Lung Adenocarcinoma Cells.

This work reports a straightforward regioselective synthetic methodology to prepare α-aminophosphine oxides and phosphonates through the addition of oxygen and sulfur nucleophiles to the C-N double bond of 2H-azirine derivatives. Determined by the nature of the nucleophile, different α-aminophosphorus compounds may be obtained. For instance, aliphatic alcohols such as methanol or ethanol afford α-aminophosphine oxide and phosphonate acetals after N-C3 ring opening of the intermediate aziridine. However, addition of 2,2,2-trifluoroethanol, phenols, substituted benzenthiols or ethanethiol to 2H-azirine phosphine oxides or phosphonates yields allylic α-aminophosphine oxides and phosphonates in good to high general yields. In some cases, the intermediate aziridine attained by the nucleophilic addition of O- or S-nucleophiles to the starting 2H-azirine may be isolated and characterized before ring opening. Additionally, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and non-malignant cells (MCR-5) was also screened. Some α-aminophosphorus derivatives exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over non-malignant cells (MCR-5) has been detected in almost all compounds tested.

Direct transfer of tri- and di-fluoroethanol units enabled by radical activation of organosilicon reagents.

Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z'.

Folding and structural polymorphism of p53 C-terminal domain: One peptide with many conformations.

Proteins of the p53 family are best known for their role in the regulation of cell cycle. The p53 protein, as a model system, has been extensively explored in numerous cancer-related studies. The C-terminal domain (CTD) of p53 is an intrinsically disordered region that gains multiple different conformations at interaction with different binding partners. However, the impact of the surrounding environment on the structural preference of p53-CTD is not known. We investigated the impact of the surrounding environment on the conformational behavior and folding of p53-CTD. Although the entire CTD is predicted as a highly disordered region by several commonly used disorder predictors, based on the secondary structure prediction, we find that a part of the CTD sequence (residues 380-388) is "confused", being predicted to shuffle between the irregular, α-helical and ß-strand structures. First time, we are observing the effect of folding-induced organic solvents, trifluoroethanol and methanol, on the conformation of CTD. Water-miscible organic solvents exert hydrophobic interactions, which are major driving force to trigger structural changes in CTD. By lowering the solution dielectric constant, organic solvents can also strengthen electrostatic interactions. We have also performed Replica Exchange Molecular Dynamic (REMD) simulations for enhanced conformation sampling of the peptide. These simulation studies have also provided detailed insight into the peculiarities of this peptide, explaining its folding behavior in the presence of methanol. We consider that these hydrophobic interactions may have important roles for function-related structural changes of this disordered region.

Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C-Terminal Domain of Histone H1.0.

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T118 -H1.0 (T118 PKK motif) and T140 -H1.0 (T140 PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T118 -H1.0 and pT118 -H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104-115 and a short α/310 helix (residues 119-123), that are almost perpendicular in T118 -H1.0 but have a poorly defined orientation in pT118 -H1.0. Peptides T140 -H1.0 and pT140 -H1.0 form very similar α helices between residues 141-147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT118 -H1.0 and pT140 -H1.0 has pKa values below the intrinsic values, which can be explained by non-specific charge-charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT140 pKa being closer to the intrinsic pKa value than the pT118 pKa . Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.

Biomacromolecules enabled dendrite-free lithium metal battery and its origin revealed by cryo-electron microscopy.

Metallic lithium anodes are highly promising for revolutionizing current rechargeable batteries because of their ultrahigh energy density. However, the application of lithium metal batteries is considerably impeded by lithium dendrite growth. Here, a biomacromolecule matrix obtained from the natural membrane of eggshell is introduced to control lithium growth and the mechanism is motivated by how living organisms regulate the orientation of inorganic crystals in biomineralization. Specifically, cryo-electron microscopy is utilized to probe the structure of lithium at the atomic level. The dendrites growing along the preferred < 111 > crystallographic orientation are greatly suppressed in the presence of the biomacromolecule. Furthermore, the naturally soluble chemical species in the biomacromolecules can participate in the formation of solid electrolyte interphase upon cycling, thus effectively homogenizing the lithium deposition. The lithium anodes employing bioinspired design exhibit enhanced cycling capability. This work sheds light on identifying substantial challenges in lithium anodes for developing advanced batteries.

Inhibiting Human Calcitonin Fibril Formation with Its Most Relevant Aggregation-Resistant Analog.

The most common obstacles to the development of therapeutic polypeptides are peptide stability and aggregation. Human calcitonin (hCT) is a 32-residue hormone polypeptide secreted from the C-cells of the thyroid gland and is responsible for calcium and phosphate regulation in the blood. hCT reduces calcium levels by inhibiting the activity of osteoclasts, which are bone cells that are mainly responsible for breaking down the bone tissue or decreasing the resorption of calcium from the kidneys. Thus, calcitonin injection has been used to treat osteoporosis and Paget's disease of bone. hCT is an aggregation-prone peptide with a high tendency to form amyloid fibrils. As a result, salmon calcitonin (sCT), which is different from hCT at 16-residue positions and has a lower propensity to aggregate, has been chosen as a clinical substitute for hCT. However, significant side effects, including immune reactions, have been shown with the use of sCT injection. In this study, we found that two residues, Tyr-12 and Asn-17, play key roles in inducing the fibrillization of hCT. Double mutation of hCT at these two crucial sites could greatly enhance its resistance to aggregation and provide a peptide-based inhibitor to prevent amyloid formation by hCT. Double-mutated hCT retains its ability to interact with its receptor in vivo. These findings suggest that this variant of hCT would serve as a valuable therapeutic alternative to sCT.

Examination of Trifluoroethanol Interactions with Trp-Cage in Trifluoroethanol-Water at 298 K through Molecular Dynamics Simulations and Intermolecular Nuclear Overhauser Effects.

Molecular dynamics simulations of the protein model Trp-cage in 42% trifluoroethanol (TFE)-water at 298 K have been carried out with the goal of exploring peptide hydrogen-solvent fluorine nuclear spin cross-relaxation. The TFE5 model of TFE developed in a previous work was used with the TIP5P-Ew model of water. System densities and component translational diffusion coefficients predicted by the simulations were within 20% of the experimental values. Consideration of the calculated relative amounts of TFE and water surrounding the hydrogens of Trp-cage indicated that the composition of the solvent mixture beyond ∼1.5 nm from the van der Waals surface of the peptide is close to the composition of the bulk solvent, but as observed by others, TFE accumulates preferentially near the peptide surface. In the simulations, both TFE and water molecules make contacts with the peptide surface; water molecules predominate in contacts with the peptide backbone atoms and TFE molecules generally preferentially interact with side chains. Translational diffusion of solvent molecules appears to be slowed near the surface of the peptide. Depending on the location in the structure, TFE molecules form complexes with the peptide that may persist for up to ∼7 ns. Many of the peptide spin-solvent fluorine cross-relaxation parameters (ΣHF) for which experimental values are available are reasonably well-predicted from the simulations. However, the calculated ΣHF values were too small for some hydrogens of the 6Trp indole ring and the amino acid hydrogens near this residue in the native structure, whereas ΣHF values for hydrogens on the side chains of 1Asn, 4Ile, and 7Leu are too large. In 42% TFE-water, persistent conformations of Trp-cage are found, which differ from the conformation found in water by the orientation of the 3Tyr ring.