RESUMEN
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.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Trifluoroetanol/química , Trifluoroetanol/farmacología , Estructura Secundaria de Proteína , Dicroismo Circular , Aminoácidos , Pliegue de Proteína , Desnaturalización ProteicaRESUMEN
Alcohols and urea are widely used as effective protein denaturants. Among monohydric alcohols, 2,2,2-trifluoroethanol (TFE) has large cosolvent effects as a helix stabilizer in proteins. In contrast, urea efficiently denatures ordered native structures, including helices, into coils. These opposing cosolvent effects of TFE and urea are well known, even though both preferentially bind to proteins; however, the underlying molecular mechanism remains controversial. Cosolvent-dependent relative stability between native and denatured states is rigorously related to the difference in preferential binding parameters (PBPs) between these states. In this study, GCN4-p1 with two-stranded coiled coil helices was employed as a model protein, and molecular dynamics simulations for the helix dimer and isolated coil were conducted in aqueous solutions with 2 M TFE and urea. As 2 M cosolvent aqueous solutions did not exhibit clustering of cosolvent molecules, we were able to directly investigate the molecular origin of the excess PBP without considering the enhancement effect of PBPs arising from the concentration fluctuations. The calculated excess PBPs of TFE for the helices and those of urea for the coils were consistent with experimentally observed stabilization of helix by TFE and that of coil by urea. The former was caused by electrostatic interactions between TFE and side chains of the helices, while the latter was attributed to both electrostatic and dispersion interactions between urea and the main chains. Unexpectedly, reverse-micelle-like orientations of TFE molecules strengthened the electrostatic interactions between TFE and the side chains, resulting in strengthening of TFE solvation.
Asunto(s)
Pliegue de Proteína , Trifluoroetanol , Trifluoroetanol/farmacología , Urea/farmacología , Etanol , Agua , Dicroismo CircularRESUMEN
The cosolvent 2,2,2-trifluoroethanol (TFE) is often used to mimic protein desiccation. We assessed the effects of TFE on cytosolic abundant heat soluble protein D (CAHS D) from tardigrades. CAHS D is a member of a unique protein class that is necessary and sufficient for tardigrades to survive desiccation. We find that the response of CAHS D to TFE depends on the concentration of both species. Dilute CAHS D remains soluble and, like most proteins exposed to TFE, gains α-helix. More concentrated solutions of CAHS D in TFE accumulate ß-sheet, driving both gel formation and aggregation. At even higher TFE and CAHS D concentrations, samples phase separate without aggregation or increases in helix. Our observations show the importance of considering protein concentration when using TFE.
Asunto(s)
Tardigrada , Trifluoroetanol , Animales , Trifluoroetanol/farmacología , Desecación , Estructura Secundaria de Proteína , Proteínas/metabolismo , Tardigrada/metabolismo , Dicroismo CircularRESUMEN
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.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Solventes , Proteínas Intrínsecamente Desordenadas/genética , Estructura Secundaria de Proteína , Enlace de Hidrógeno , Secuencia de Aminoácidos , Saccharomyces cerevisiae , Dicroismo Circular , Trifluoroetanol/farmacología , Pliegue de ProteínaRESUMEN
Despite the rich knowledge of the influence of 2,2,2-trifluoroethanol (TFE) on the structure and conformation of peptides and proteins, the mode(s) of TFE-protein interactions and the mechanism by which TFE reversibly denatures a globular protein remain elusive. This study systematically examines TFE-induced equilibrium transition curves for six paradigmatic globular proteins by using basic fluorescence and circular dichroism measurements under neutral pH conditions. The results are remarkably simple. Low TFE invariably unfolds the tertiary structure of all proteins to produce the obligate intermediate (I) which retains nearly all of native-state secondary structure, but enables the formation of extra α-helices as the level of TFE is raised higher. Inspection of the transitions at once reveals that the tertiary structure unfolding is always a distinct process, necessitating the inclusion of at least one obligate intermediate in the TFE-induced protein denaturation. It appears that the intermediate in the minimal unfolding mechanism NâIâD somehow acquires higher α-helical propensity to generate α-helices in excess of that in the native state to produce the denatured state (D), also called the TFE state. The low TFE-populated intermediate I may be called a universal intermediate by virtue of its α-helical propensity. Contrary to many earlier suggestions, this study dismisses molten globule (MG)-like attribute of I or D.
Asunto(s)
Trifluoroetanol , Naftalenosulfonatos de Anilina/química , Naftalenosulfonatos de Anilina/metabolismo , Dicroismo Circular , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Trifluoroetanol/farmacologíaRESUMEN
We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.
Asunto(s)
Membrana Celular/efectos de los fármacos , Citotoxinas/metabolismo , Citotoxinas/farmacología , Lactalbúmina/metabolismo , Lactalbúmina/farmacología , Animales , Bovinos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Dicroismo Circular , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Trifluoroetanol/metabolismo , Trifluoroetanol/farmacología , Células U937RESUMEN
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.
Asunto(s)
Agregado de Proteínas/efectos de los fármacos , Desplegamiento Proteico/efectos de los fármacos , Superóxido Dismutasa/química , Trifluoroetanol/farmacología , Humanos , Espectrometría de Masas , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Huella de Proteína , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading microorganisms. Although AMPs are known to act on bacterial membranes and increase membrane permeability, the action mechanism of most AMPs still remains unclear. In this report, we found that the TP4 peptides from Nile tilapia anchored on E. coli cells and enabled them permeable to SYTOX Green in few minutes after TP4 addition. TP4 peptides existed in small dots either on live or glutaraldehyde-fixed cells. TP4 peptides were driven into oligomers either in soluble or insoluble form by a membrane-mimicking anionic surfactant, sarkosyl, depending on the concentrations employed. The binding forces among TP4 components were mediated through hydrophobic interaction. The soluble oligomers were negatively charged on surface, while the insoluble oligomers could be fused with each other or piled on existing particles to form larger particles with diameters 0.1 to 20 µm by hydrophobic interactions. Interestingly, the morphology and solubility of TP4 particles changed with the concentration of exogenous sarkosyl or trifluoroethanol. The TP4 peptides were assembled into oligomers on or in bacterial membrane. This study provides direct evidence and a model for the oligomerization and insertion of AMPs into bacterial membrane before entering into cytosol.
Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Proteínas de Peces/química , Tensoactivos/farmacología , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Transporte Biológico , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cíclidos/fisiología , Escherichia coli/metabolismo , Proteínas de Peces/aislamiento & purificación , Proteínas de Peces/farmacología , Colorantes Fluorescentes/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Compuestos Orgánicos/metabolismo , Unión Proteica , Multimerización de Proteína , Sarcosina/análogos & derivados , Sarcosina/farmacología , Electricidad Estática , Trifluoroetanol/farmacologíaRESUMEN
Hyperactivated macrophages play a key role in the initiation and perpetuation of mucosal inflammation in Crohn's disease (CD). Increasing evidence suggests that the basic helix-loop-helix (bHLH) repressor Twist1 can suppress activation of nuclear factor-κB (NF-κB) and the subsequent production of TNF-α, which are both essential elements of macrophage activation. Thus, developing novel therapeutic strategies to enhance Twist1 expression and to inhibit macrophage activation may be beneficial for CD treatment. In the present study, a series of trifluoroethyl thiazolo[3,2-b][1,2,4]triazole derivatives were used to investigate their potential anti-inflammatory activities and the underlying mechanism. In a biological activity screen, compound 7# (Thiazolo[3,2-b][1,2,4]triazole-5-methanamine, 6-phenyl-α-(trifluoromethyl)-, (αR)-, TT-TFM) suppressed the activation of macrophages. Consistent with the in vitro data, TT-TFM protected against 2,4,6-trinitrobenzene sulfonic acid (TNBS), dextran sulfate sodium (DSS)-induced acute colitis and IL-10 knockout (KO) chronic colitis, as judged by body weight changes and colonic pathological damage. A mechanistic study based on microarray analysis and gene interference experiments indicated that TT-TFM exerted anti-inflammatory effects by enhancing Twist1 expression and subsequently blocking the NF-κB/TNF-α pathway. In addition, pretreatment with lentiviruses encoding shRNA targeting Twist1 could abolish the therapeutic effect of TT-TFM in TNBS colitis. Ultimately, TT-TFM showed anti-colitis activity by reducing NF-κB activation and the TNF-α level by promoting Twist1 expression; thus, TT-TFM may offer a therapeutic strategy for CD patients.
Asunto(s)
Colitis/metabolismo , Activación de Macrófagos/efectos de los fármacos , Proteínas Nucleares/biosíntesis , Transducción de Señal/fisiología , Triazoles/química , Triazoles/uso terapéutico , Proteína 1 Relacionada con Twist/biosíntesis , Animales , Células Cultivadas , Colitis/tratamiento farmacológico , Femenino , Activación de Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Proteínas Nucleares/agonistas , Transducción de Señal/efectos de los fármacos , Triazoles/farmacología , Trifluoroetanol/química , Trifluoroetanol/farmacología , Trifluoroetanol/uso terapéutico , Proteína 1 Relacionada con Twist/agonistasRESUMEN
The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.
Asunto(s)
Amiloide/efectos de los fármacos , Ataxina-3/metabolismo , Agregado de Proteínas/efectos de los fármacos , Proteínas Represoras/metabolismo , Trifluoroetanol/farmacología , Ataxina-3/química , Dicroismo Circular , Humanos , Conformación Molecular , Simulación de Dinámica Molecular , Péptidos/metabolismo , Conformación Proteica/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Proteínas Represoras/químicaRESUMEN
The Parkinson's protein alpha-synuclein binds to synaptic vesicles in vivo and adopts a highly extended helical conformation when binding to lipid vesicles in vitro. High-resolution structural analysis of alpha-synuclein bound to small lipid or detergent micelles revealed two helices connected by a non-helical linker, but corresponding studies of the vesicle-bound extended-helix state are hampered by the size and heterogeneity of the protein-vesicle complex. Here we employ fluorinated alcohols (FAs) to induce a highly helical aggregation-resistant state of alpha-synuclein in solution that resembles the vesicle-bound extended-helix state but is amenable to characterization using high-resolution solution-state NMR. Analysis of chemical shift, NOE, coupling constant, PRE and relaxation measurements shows that the lipid-binding domain of alpha-synuclein in FA solutions indeed adopts a single continuous helix and that the ends of this helix do not come into detectable proximity to each other. The helix is well ordered in the center, but features an increase in fast internal motions suggestive of helix fraying approaching the termini. The central region of the helix exhibits slower time scale motions that likely result from flexing of the highly anisotropic structure. Importantly, weak or missing short- and intermediate-range NOEs in the region corresponding to the non-helical linker of micelle-bound alpha-synuclein indicate that the helical structure in this region of the protein is intrinsically unstable. This suggests that conversion of alpha-synuclein from the extended-helix to the broken-helix state represents a functionally relevant structural transition.
Asunto(s)
Alcoholes/farmacología , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Enfermedad de Parkinson/metabolismo , Propanoles/farmacología , Agregado de Proteínas/efectos de los fármacos , Estructura Secundaria de Proteína , Trifluoroetanol/farmacologíaRESUMEN
Misfolding and aggregation of Cu, Zn Superoxide dismutase (SOD1) is involved in the neurodegenerative disease, amyotrophic lateral sclerosis. Many studies have shown that metal-depleted, monomeric form of SOD1 displays substantial local unfolding dynamics and is the precursor for aggregation. Here, we have studied the structure and dynamics of different apo monomeric SOD1 variants associated with unfolding and aggregation in aqueous trifluoroethanol (TFE) through experiments and simulation. TFE induces partially unfolded ß-sheet-rich extended conformations in these SOD1 variants, which subsequently develops aggregates with fibril-like characteristics. Fibrillation was achieved more easily in disulfide-reduced monomeric SOD1 when compared with wild-type and mutant monomeric SOD1. At higher concentrations of TFE, a native-like structure with the increase in α-helical content was observed. The molecular dynamics simulation results illustrate distinct structural dynamics for different regions of SOD1 variants and show uniform local unfolding of ß-strands. The strands protected by the zinc-binding and electrostatic loops were found to unfold first in 20% (v/v) TFE, leading to a partial unfolding of ß-strands 4, 5, and 6 which are prone to aggregation. Our results thus shed light on the role of local unfolding and conformational dynamics in SOD1 misfolding and aggregation.
Asunto(s)
Simulación de Dinámica Molecular , Mutación , Pliegue de Proteína , Estructura Secundaria de Proteína , Superóxido Dismutasa-1/química , Trifluoroetanol/farmacología , Estabilidad de Enzimas , Humanos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Alpha-2-macroglobulin (α2M), a pan-proteinase inhibitor, inhibits a variety of endogenous and exogenous proteinases and constitutes an important part of body's innate defense system. In the present study, we explored how trifluoroethanol (TFE) may modulate the structure, antiproteinase activity and aggregation of α2M. TFE was sequentially added over a range of 0-20% (v/v) and the effects induced were studied by activity assay, intrinsic fluorescence, ANS fluorescence, circular dichroism, turbidity assay, Rayleigh scattering measurement and ThT fluorescence measurement. Decrease in activity and increase in fluorescence intensity of α2M upon addition of TFE shows structural deviation from the native structure and suggests aggregation of protein upon solvent addition. Increase in turbidity and Rayleigh scattering of modified α2M confirms the formation of aggregates. Insignificant ThT fluorescence intensity of TFE treated α2M is indicative of amorphous or non-amyloid aggregation. Further, circular dichroism results indicate the changes in secondary structure of native α2M as negative ellipticity decreased on addition of the polar solvent to the inhibitor. The turbidometric analysis, Rayleigh scattering, ThT fluorescence intensity of modified α2M suggests that the protein might be driven towards non-amyloid or amorphous aggregation. Our studies provide important mechanistic insight how α2M undergoes conformational and functional changes when exposed to TFE.
Asunto(s)
alfa 2-Macroglobulinas Asociadas al Embarazo/química , Agregado de Proteínas/efectos de los fármacos , Trifluoroetanol/farmacología , Animales , Péptido Hidrolasas/metabolismo , alfa 2-Macroglobulinas Asociadas al Embarazo/metabolismo , Conformación Proteica/efectos de los fármacos , Ovinos , Solventes/farmacologíaRESUMEN
Glucoamylase (EC 3.2.1.3) from Aspergillus niger possesses 31% α-helix, 36% ß structure and rest aperiodic structure. A transition of glucoamylase structure in the presence of varying concentrations of glyoxal (GO) and trifluoroethanol (TFE) was studied by using multi-methodological approaches. At 20% GO, glucoamylase exists as molten globule state as evident by high tryptophan and ANS fluorescence, retention of secondary structure and loss of native tertiary structure. This state precedes the onset of the aggregation process and maximum is achieved at the highest concentration i.e. at 90% of GO. In parallel study TFE, on increasing concentration up to 25% induces secondary structure transformation leading to accumulation of intermolecular ß sheets, altered tryptophan environment, high ANS and ThT fluorescence resulting in the formation of glucoamylase aggregates. Isothermal titration calorimetric curve is sigmoidal, indicating the weak binding of GO/TFE and glucoamylase. TEM studies showed that glucoamylase exists as globular and amorphous aggregates at 90% glyoxal and 25% TFE respectively. Further, TFE at 70% causes inhibition of enzyme aggregates; the majority of secondary structures observed at this concentration are α helices. Alpha helices being the main key player relocates glucoamylase native environment as evident by CD, FTIR and TEM. Hence induction of ß sheet promotes protein aggregation and α helices inhibits protein aggregation.
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/química , Multimerización de Proteína , Aspergillus niger/enzimología , Relación Dosis-Respuesta a Droga , Glucano 1,4-alfa-Glucosidasa/metabolismo , Glioxal/metabolismo , Glioxal/farmacología , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Conformación Proteica en Hélice alfa/efectos de los fármacos , Conformación Proteica en Lámina beta/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Trifluoroetanol/farmacologíaRESUMEN
The conversion of proteins into amyloid fibrils and other amyloid-like aggregates is closely connected to the onset of a series of age-related pathologies. Upon changes in environmental conditions, amyloid-like aggregates may also undergo disassembly into oligomeric aggregates, the latter being recognized as key effectors in toxicity. This indicates new possible routes for in vivo accumulation of toxic species. In the light of the recognized implication of α-Synuclein (αSN) in Parkinson's disease, we present an experimental study on supramolecular assembly of αSN with a focus on stability and disassembly paths of such supramolecular aggregate species. Using spectroscopic techniques, two-photon microscopy, small-angle X-ray scattering and atomic force microscopy, we report evidences on how the stability of αSN amyloid-like aggregates can be altered by changing solution conditions. We show that amyloid-like aggregate formation can be induced at high temperature in the presence of trifluoroethanol (TFE). Moreover, sudden disassembly or further structural reorganisation toward higher hierarchical species can be induced by varying TFE concentration. Our results may contribute in deciphering fundamental mechanisms and interactions underlying supramolecular clustering/dissolution of αSN oligomers in cells.
Asunto(s)
Agregado de Proteínas/efectos de los fármacos , Trifluoroetanol/farmacología , alfa-Sinucleína/química , Amiloide/química , Humanos , Estabilidad Proteica , Análisis Espectral , Temperatura , alfa-Sinucleína/efectos de los fármacosRESUMEN
α-Crystallin, a member of small heat shock proteins, is the major structural protein within the eye lens and is believed to play an exceptional role in the stability of lens proteins and its transparency. In the current manuscript, we have investigated the effect of an organic solvent, trifluoroethanol (TFE), on the structure and function of α-crystallin isolated from camel eye lens. Incubation of this protein with TFE changed the secondary and tertiary structures, which resulted in the aggregation of α-crystallin as evidenced by intrinsic fluorescence, Rayleigh's scattering, Thioflavin T assay, and circular dichroism spectroscopic studies. The treatment with different concentrations of TFE led to increased exposure of hydrophobic domains of α-crystallin, which was observed by 8-anilino 1-napthalene sulfonic acid extrinsic fluorescence assay. These results clearly indicate that TFE induced significant changes in the secondary and tertiary structures of α-crystallin, leading to aggregation and amyloid formation. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay established the cytotoxicity of the aggregated α-crystallin towards HepG2 cell lines through reactive oxygen species production. In conclusion, α-crystallin protein was found to be susceptible to conformational changes by TFE, suggesting that α-crystallin, although basically acting like a heat shock protein and functionally displaying chaperone-like activity, might capitulate to change in lens environment induced by diseased conditions or age-related changes, resulting in cataract formation.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Trifluoroetanol/farmacología , alfa-Cristalinas/química , alfa-Cristalinas/efectos de los fármacos , Amiloide/efectos de los fármacos , Animales , Camelus , Dicroismo Circular , Células Hep G2 , Humanos , Modelos Moleculares , Agregado de Proteínas , Pliegue de Proteína , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , alfa-Cristalinas/aislamiento & purificaciónRESUMEN
Southern bean mosaic virus (SBMV) RNA purified from infected plants was used for cloning the viral genome-linked protein (VPg) and was subsequently expressed in Escherichia coli. Circular dichroism (CD), dynamic light scattering (DLS) and saturation transfer difference (STD) by nuclear magnetic resonance (NMR) measurements were employed to determine the degree of monodispersity and to investigate the conformational changes in the absence and presence of trifluoroethanol (TFE) which indicated increased helical content with increasing concentration of TFE. 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as a probe to compare the unfolding regions of the protein before and after addition of TFE. The results indicated that although the TFE concentration influences VPg folding, it does not play a role in nucleotide binding and that the local solvent hydrophobicity causes significant conformational changes.
Asunto(s)
Fabaceae/virología , Virus de Plantas/genética , Virus de Plantas/metabolismo , Trifluoroetanol/metabolismo , Trifluoroetanol/farmacología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Expresión Génica , Histidina , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Unión Proteica , Conformación Proteica/efectos de los fármacos , Proteínas no Estructurales Virales/químicaRESUMEN
The solution structure of enterocin HF (EntHF), a class IIa bacteriocin of 43 amino acids produced by Enterococcus faecium M3K31, was evaluated by CD and NMR spectroscopy. Purified EntHF was unstructured in water, but CD analysis supports that EntHF adopts an α-helical conformation when exposed to increasing concentrations of trifluoroethanol. Furthermore, NMR spectroscopy indicates that this bacteriocin adopts an antiparallel ß-sheet structure in the N-terminal region (residues 1-17), followed by a well-defined central α-helix (residues 19-30) and a more disordered C-terminal end (residues 31-43). EntHF could be structurally organized into three flexible regions that might act in a coordinated manner. This is in agreement with the absence of long-range nuclear Overhauser effect signals between the ß-sheet domain and the C-terminal end of the bacteriocin. The 3D structure recorded for EntHF fits emerging facts regarding target recognition and mode of action of class IIa bacteriocins.
Asunto(s)
Bacteriocinas/química , Enterococcus faecium/metabolismo , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Soluciones , Trifluoroetanol/farmacologíaRESUMEN
Trifluoroethanol (TFE) mimics the membrane environments as it simulates the hydrophobic environment and better stabilizes the secondary structures in peptides owing to its hydrophobicity and hydrogen bond-forming properties. Its dielectric constant approximates that of the interior of proteins and is one-third of that of water. Human serum albumin (HSA) is a biological transporter. The effect of TFE on HSA gives the clue about the conformational changes taking place in HSA on transport of ligands across the biological membranes. At 25% (v/v) and 60% (v/v) TFE, HSA exhibits non-native ß-sheet, altered tryptophan fluorescence, exposed hydrophobic clusters, increased thioflavin T fluorescence and prominent red shifted Congo red absorbance, and large hydrodynamic radii suggesting the aggregate formation. Isothermal titration calorimetric results indicate weak binding of TFE and HSA. This suggests that solvent-mediated effects dominate the interaction of TFE and HSA. TEM confirmed prefibrillar at 25% (v/v) and fibrillar aggregates at 60% (v/v) TFE. Comet assay of prefibrillar aggregates showed DNA damage causing cell necrosis hence confirming cytotoxic nature. On increasing concentration of TFE to 80% (v/v), HSA showed retention of native-like secondary structure, increased Trp and ANS fluorescence, a transition from ß-sheet to α-helix. Thus, TFE at high concentration possess anti- aggregating potency.
Asunto(s)
Anestésicos/química , Agregado de Proteínas , Albúmina Sérica/química , Trifluoroetanol/química , Anestésicos/farmacología , Benzotiazoles , Ensayo Cometa , Rojo Congo , Colorantes Fluorescentes , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Linfocitos/química , Linfocitos/citología , Linfocitos/efectos de los fármacos , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Cultivo Primario de Células , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Soluciones , Tiazoles , Trifluoroetanol/farmacologíaRESUMEN
The bacterial mechanosensitive channel of small conductance (MscS) plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. The functional characteristics of MscS have been extensively studied using liposomal reconstitution. This is a widely used experimental paradigm and is particularly important for mechanosensitive channels as channel activity can be probed free from cytoskeletal influence. A perpetual issue encountered using this paradigm is unknown channel orientation. Here we examine the orientation of MscS in liposomes formed using 2 ion channel reconstitution methods employing the powerful combination of patch clamp electrophysiology, confocal microscopy, and continuum mechanics simulation. Using the previously determined electrophysiological and pharmacological properties of MscS, we were able to determine that in liposomes, independent of lipid composition, MscS adopts the same orientation seen in native membranes. These results strongly support the idea that these specific methods result in uniform incorporation of membrane ion channels and caution against making assumptions about mechanosensitive channel orientation using the stimulus type alone.