RESUMO
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.
Assuntos
Álcoois , Meliteno , Meliteno/química , Solventes/química , Álcoois/química , Peptídeos/química , Proteínas/química , Água/química , Trifluoretanol/químicaRESUMO
Saccharomyces cerevisiae IA3 is a 68 amino acid peptide inhibitor of yeast proteinase A (YPRA) characterized as a random coil when in solution, folding into an N-terminal amphipathic alpha helix for residues 2-32 when bound to YPRA, with residues 33-68 unresolved in the crystal complex. Circular dichroism (CD) spectroscopy results show that amino acid substitutions that remove hydrogen-bonding interactions observed within the hydrophilic face of the N-terminal domain (NTD) of IA3-YPRA crystal complex reduce the 2,2,2-trifluoroethanol (TFE)-induced helical transition in solution. Although nearly all substitutions decreased TFE-induced helicity compared to wild-type (WT), each construct did retain helical character in the presence of 30% (v/v) TFE and retained disorder in the absence of TFE. The NTDs of 8 different Saccharomyces species have nearly identical amino acid sequences, indicating that the NTD of IA3 may be highly evolved to adopt a helical fold when bound to YPRA and in the presence of TFE but remain unstructured in solution. Only one natural amino acid substitution explored within the solvent-exposed face of the NTD of IA3 induced TFE-helicity greater than the WT sequence. However, chemical modification of a cysteine by a nitroxide spin label that contains an acetamide side chain did enhance TFE-induced helicity. This finding suggests that non-natural amino acids that can increase hydrogen bonding or alter hydration through side-chain interactions may be important to consider when rationally designing intrinsically disordered proteins (IDPs) with varied biotechnological applications.
Assuntos
Proteínas Intrinsicamente Desordenadas , Solventes , Proteínas Intrinsicamente Desordenadas/genética , Estrutura Secundária de Proteína , Ligação de Hidrogênio , Sequência de Aminoácidos , Saccharomyces cerevisiae , Dicroísmo Circular , Trifluoretanol/farmacologia , Dobramento de ProteínaRESUMO
Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol-water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol-water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains-transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP.
Assuntos
Trifluoretanol , Água , Ressonância Magnética Nuclear Biomolecular , Proteínas de Membrana , Lipídeos/químicaRESUMO
Although rarely used in nature, fluorine has emerged as an important elemental ingredient in the design of proteins with altered folding, stability, oligomerization propensities, and bioactivity. Adding to the molecular modification toolbox, here we report the ability of privileged perfluorinated amphiphiles to noncovalently decorate proteins to alter their conformational plasticity and potentiate their dispersion into fluorous phases. Employing a complementary suite of biophysical, in-silico and in-vitro approaches, we establish structure-activity relationships defining these phenomena and investigate their impact on protein structural dynamics and intracellular trafficking. Notably, we show that the lead compound, perfluorononanoic acid, is 106 times more potent in inducing non-native protein secondary structure in select proteins than is the well-known helix inducer trifluoroethanol, and also significantly enhances the cellular uptake of complexed proteins. These findings could advance the rational design of fluorinated proteins, inform on potential modes of toxicity for perfluoroalkyl substances, and guide the development of fluorine-modified biologics with desirable functional properties for drug discovery and delivery applications.
Assuntos
Flúor , Proteínas , Flúor/química , Proteínas/química , Estrutura Secundária de Proteína , TrifluoretanolRESUMO
Whereas iron catalysts usually promote catalyzed C3-alkylation of indole derivatives via a borrowing-hydrogen methodology using alcohols as the electrophilic partners, this contribution shows how to switch the selectivity towards N-alkylation. Thus, starting from indoline derivatives, N-alkylation was efficiently performed using a tricarbonyl(cyclopentadienone) iron complex as the catalyst in trifluoroethanol in the presence of alcohols leading to the corresponding N-alkylated indoline derivatives in 31-99 % yields (28â examples). The one-pot, two-step strategy for the selective N-alkylation of indolines is completed by an oxidation to give the corresponding N-alkylated indoles in 31-90 % yields (15â examples). This unprecedented oxidation methodology involves an iron salt catalyst associated with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) and a stoichiometric amount of t-BuOOH at room temperature.
Assuntos
Ferro , Trifluoretanol , Álcoois , Alquilação , Catálise , Hidrogênio , IndóisRESUMO
When formic acid and 2,2,2-trifluoroethanol are co-expanded through a slit nozzle into vacuum, a single dominant, hinge-like 1 : 1 complex is formed in significant amounts and its two OH stretching fundamentals separated by 100 cm-1 can be unambiguously assigned by a combination of infrared absorption and Raman scattering. Quantum chemical calculations at different levels reproduce this finding in a satisfactory way and suggest that in-phase (Raman-sensitive and lower wavenumber) OH stretch excitation more or less along the concerted degenerate proton transfer coordinate in the hydrogen-bonded ring stays below the barrier for this concerted exchange. Anharmonic calculations indicate only weak intensity sharing with dark states coming into reach due to the hydrogen bond downshift of the OH stretching vibration. This well-behaved system sets the stage for acid combinations with more basic alcohols, where the in-phase OH stretching vibration is more difficult to detect, possibly due to fast intra-complex vibrational dynamics. It thus provides a benchmark point from which one can explore the evolution of vibrational resonances when the acidic proton meets a more electron-rich alcoholic oxygen.
Assuntos
Prótons , Vibração , Trifluoretanol , Ligação de Hidrogênio , FormiatosRESUMO
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 OHâ¯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.
Assuntos
Halogenação , Trifluoretanol , Ligação de Hidrogênio , Espectrofotometria Infravermelho/métodos , Trifluoretanol/químicaRESUMO
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.
Assuntos
COVID-19 , Influenza Humana , Humanos , Betainfluenzavirus , Coronavírus da Síndrome Respiratória do Oriente Médio , Simulação de Dinâmica Molecular , Peptídeos/química , Dobramento de Proteína , Estrutura Secundária de Proteína , SARS-CoV-2 , Solventes , Trifluoretanol/química , Proteínas ViroporinasRESUMO
The 1:1 adduct of 1-phenyl-2,2,2-trifluoroethanol (PhTFE), a chiral fluoroalcohol, with water was investigated using chirped pulse Fourier transform microwave spectroscopy and computational methods. While PhTFE itself was predicted to have three minima, I (gauche+), II (trans), and III (gauche-), only I and II were stable and only I was observed experimentally. A systematic search of the PhTFE···H2O conformational landscape identified 110 stable minima, 14 of which are within a 15 kJ mol-1 energy window. Rotational spectra of the two PhTFE···H2O conformers along with several deuterium and 18O isotopologues were assigned, and the isotopic data were used to verify the corresponding structures. In the two observed monohydrate conformers, one contains PhTFE I where the water subunit is inserted into the existing intramolecular OH···F contact of I, and the binary adduct is stabilized by two intermolecular contacts: OH···OW and HW···F, whereas the other contains PhTFE II where the water subunit interacts with both the alcohol hydrogen and phenyl ring of II, demonstrating that interaction with water sufficiently stabilizes II for its observation in a jet expansion. Interestingly, the predicted electric dipole moment components at the identified minima deviate considerably from the experimental ones. Such deviations were analyzed in terms of dynamic effects associated with the large amplitude motions of the unbound HW. In addition, tunnelling effects associated with the exchange of the bonded and nonbonded HW were also discussed.
Assuntos
Trifluoretanol , Água , Deutério , Conformação Molecular , Trifluoretanol/análogos & derivadosRESUMO
We report an analysis of high-resolution quasielastic neutron scattering spectra from Myelin Basic Protein (MBP) in solution, comparing the spectra at three different temperatures (283, 303, and 323 K) for a pure D2O buffer and a mixture of D2O buffer with 30% of deuterated trifluoroethanol (TFE). Accompanying experiments with dynamic light scattering and Circular Dichroism (CD) spectroscopy have been performed to obtain, respectively, the global diffusion constant and the secondary structure content of the molecule for both buffers as a function of temperature. Modeling the decay of the neutron intermediate scattering function by the Mittag-Leffler relaxation function, Ï(t) = Eα(-(t/τ)α) (0 < α < 1), we find that trifluoroethanol slows down the relaxation dynamics of the protein at 283 K and leads to a broader relaxation rate spectrum. This effect vanishes with increasing temperature, and at 323 K, its relaxation dynamics is identical in both solvents. These results are coherent with the data from dynamic light scattering, which show that the hydrodynamic radius of MBP in TFE-enriched solutions does not depend on temperature and is only slightly smaller compared to the pure D2O buffer, except for 283 K, where it is much reduced. In accordance with these observations, the CD spectra reveal that TFE induces essentially a partial transition from ß-strands to α-helices, but only a weak increase in the total secondary structure content, leaving about 50% of the protein unfolded. The results show that MBP is for all temperatures and in both buffers an intrinsically disordered protein and that TFE essentially induces a reduction in its hydrodynamic radius and its relaxation dynamics at low temperatures.
Assuntos
Proteína Básica da Mielina , Difração de Nêutrons , Proteína Básica da Mielina/química , Proteína Básica da Mielina/metabolismo , Nêutrons , Estrutura Secundária de Proteína , Soluções , TrifluoretanolRESUMO
A new mild and practically simple alkyne hydroarylation protocol for the synthesis of 3-(indol-3-yl)-3-(trifluoromethyl)acrylic acid esters by the reaction of indole derivatives with ethyl/methyl 4,4,4-trifluoro-3-(indol-3-yl)but-2-enoates in trifluoroethanol was developed. This method has the following advantages: no catalyst, atom economy, high yields, broad substrate scope, and large-scale synthesis. The potential application of this protocol was further demonstrated by the synthesis of a variety of CF3 -substituted synthons and a new class of (un)symmetrical 3,3'-diindolylmethanes with a quaternary carbon core that might be biologically active.
Assuntos
Alcinos , Trifluoretanol , Catálise , Indóis , Relação Estrutura-AtividadeRESUMO
Background: Hemoglobin (Hb) variants arise due to point mutations in globin chains and their pathological treatments rely heavily on the identification of the nature and location of the mutation in the globin chains. Traditional methods for diagnosis such as HPLC and electrophoresis have their own limitations. Therefore, the present study aims to develop and optimize a specific method of sample processing that could lead to improved sequence coverage and analysis of Hb variants by nano LC-MALDI MS/MS. Methods: In our study, we primarily standardized various sample processing methods such as conventional digestion with trypsin followed by 10% acetonitrile treatment, digestion with multiple proteases like trypsin, Glu-C, Lys-C, and trypsin digestion subsequent to 2,2,2 trifluoroethanol (TFE) treatment. Finally, the peptides were identified by LC-MALDI MS/MS. All of these sample processing steps were primarily tested with recombinant Hb samples. After initial optimization, we found that the TFE method was the most suitable one and the efficiency of this method was applied in Hb variant identification based on high sequence coverage. Results: We developed and optimized a method using an organic solvent TFE and heat denaturation prior to digestion, resulting in 100% sequence coverage in the ß-chains and 95% sequence coverage in the α-chains, which further helped in the identification of Hb mutations. A Hb variant protein sequence database was created to specify the search and reduce the search time. Conclusion: All of the mutations were identified using a bottom-up non-target approach. Therefore, a sensitive, robust and reproducible method was developed to identify single substitution mutations in the Hb variants from the sequence of the entire globin chains. Biological Significance: Over 330,000 infants are born annually with hemoglobinopathies and it is the major cause of morbidity and mortality in early childhood. Hb variants generally arise due to point mutation in the globin chains. There is high sequence homology between normal Hb and Hb variant chains. Due to this high homology between the two forms, identification of variants by mass spectrometry is very difficult and requires the full sequence coverage of α- and ß-chains. As such, there is a need for a suitable method that provides 100% sequence coverage of globin chains for variant analysis by mass spectrometry. Our study provides a simple, robust, and reproducible method that is suitable for LC-MALDI and provides nearly complete sequence coverage in the globin chains. This method may be used in the near future in routine diagnosis for Hb variant analysis.
Assuntos
Espectrometria de Massas em Tandem , Trifluoretanol , Pré-Escolar , Humanos , Acetonitrilas , Digestão , Hemoglobinas/metabolismo , Mutação , Peptídeos/genética , Solventes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/genéticaRESUMO
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 â¼2 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 â¼2 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.
Assuntos
Álcoois/química , Meliteno/química , Peptídeos/química , Solventes/química , Trifluoretanol/química , Sequência de Aminoácidos , Conformação Molecular , Transição de Fase , Termodinâmica , ÁguaRESUMO
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.
Assuntos
Etanol/química , Lactoglobulinas/química , Muramidase/química , Trifluoretanol/química , Água/química , Animais , Bovinos , Galinhas , Conformação Proteica , Estrutura Secundária de Proteína , Solubilidade , Espectroscopia TerahertzRESUMO
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.
Assuntos
Trato Gastrointestinal/efeitos dos fármacos , Desnaturação Proteica , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/farmacologia , Animais , Animais Lactentes , Células CACO-2 , Bovinos , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Diarreia/tratamento farmacológico , Humanos , Lactalbumina/química , Lactalbumina/farmacologia , Lactoglobulinas/química , Lactoglobulinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Trifluoretanol/químicaRESUMO
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.
Assuntos
Ésteres/química , Naproxeno/síntese química , Sais/química , Catálise , Química Farmacêutica/métodos , Esterificação , Flúor/química , Halogenação , Hidrólise , Iodo/química , Lipase/metabolismo , Espectroscopia de Ressonância Magnética , Fenóis/química , Solventes , Estereoisomerismo , Trifluoretanol/química , Difração de Raios XRESUMO
Misfolding of Cu, Zn superoxide dismutase (SOD1) variants may lead to protein aggregation and ultimately amyotrophic lateral sclerosis (ALS). The mechanism and protein conformational changes during this process are complex and remain unclear. To study SOD1 variant aggregation at the molecular level and in solution, we chemically induced aggregation of a mutant variant (G93A SOD1) with trifluoroethanol (TFE) and used both native mass spectrometry (MS) to analyze the intact protein and fast photochemical oxidation of proteins (FPOP) to characterize the structural changes induced by TFE. We found partially unfolded G93A SOD1 monomers prior to oligomerization and identified regions of the N-terminus, C-terminus, and strands ß5, ß6 accountable for the partial unfolding. We propose that exposure of hydrophobic interfaces of these unstructured regions serves as a precursor to aggregation. Our results provide a possible mechanism and molecular basis for ALS-linked SOD1 misfolding and aggregation.
Assuntos
Agregados Proteicos/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , Superóxido Dismutase/química , Trifluoretanol/farmacologia , Humanos , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Pegadas de Proteínas , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
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.
Assuntos
Histonas/química , Proteínas Intrinsicamente Desordenadas/química , Peptídeos/química , Trifluoretanol/química , Histonas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , FosforilaçãoRESUMO
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.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metanol/química , Simulação de Dinâmica Molecular , Domínios Proteicos , Dobramento de Proteína , Estrutura Terciária de Proteína , Temperatura , Trifluoretanol/químicaRESUMO
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.