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Background and Aims: Our goal was to develop an online questionnaire to survey the prevalence of suicidal behavior. Methods: We developed a questionnaire with 51 variables and proceeded with validations. Validations were performed using face validity, content validity, and construct validity. Reliability was performed by test-rest. Results: The face validity was 1.0 and the content validity was 0.91. The exploratory factor analysis got Kaiser-Meyer-Olkin = 0.86 and extracted one principal factor. The confirmatory factor analysis demonstrates root mean square error of approximation = 0.000 and comparative fit index = 1.000. The test-retest had an intraclass correlated coefficient of 0.98. Conclusion: The adequate development questionnaire was validated, and we have an instrument to survey suicide behaviors during the pandemic time. Patient or Public Contribution: The general population of Marília voluntarily responded to the questionnaire, as well as patients from the principal investigator's office.
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A single immediate reconstruction with free tissue transfer is the method of choice after major head and neck cancer (HNC) resection, but this is frequently associated with long operating hours. Considering regulatory working hour constraints, we investigated whether a two-staged reconstructive approach with temporary defect coverage by an artificial tissue substitute would be feasible. HNC patients underwent either immediate or delayed reconstruction after tumor resection. Patients with delayed reconstruction received preliminary reconstruction with an artificial tissue substitute followed by definitive microvascular reconstruction in a separate, second procedure. Of the 33 HNC patients, 13 received delayed reconstruction and 20 received immediate reconstruction. Total anesthesia time (714 vs. 1011 min; p < 0.002) and the total duration of hospital stay (34 ± 13 vs. 25 ± 6 days; p = 0.03) were longer in the delayed reconstruction group. Perioperative morbidity (p = 0.58), functional outcome (p > 0.1) and 5-year postoperative survival rank (p = 0.28) were comparable in both groups. Delayed reconstruction after HNC resection was feasible. Perioperative morbidity, functional outcome and overall survival were comparable to immediate reconstruction.
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Globally, over the next few decades, more than 2.5 billion people will suffer from hearing impairment, including profound hearing loss, and millions could potentially benefit from a cochlea implant. To date, several studies have focused on tissue trauma caused by cochlea implantation. The direct immune reaction in the inner ear after an implantation has not been well studied. Recently, therapeutic hypothermia has been found to positively influence the inflammatory reaction caused by electrode insertion trauma. The present study aimed to evaluate the hypothermic effect on the structure, numbers, function and reactivity of macrophages and microglial cells. Therefore, the distribution and activated forms of macrophages in the cochlea were evaluated in an electrode insertion trauma cochlea culture model in normothermic and mild hypothermic conditions. In 10-day-old mouse cochleae, artificial electrode insertion trauma was inflicted, and then they were cultured for 24 h at 37 °C and 32 °C. The influence of mild hypothermia on macrophages was evaluated using immunostaining of cryosections using antibodies against IBA1, F4/80, CD45 and CD163. A clear influence of mild hypothermia on the distribution of activated and non-activated forms of macrophages and monocytes in the inner ear was observed. Furthermore, these cells were located in the mesenchymal tissue in and around the cochlea, and the activated forms were found in and around the spiral ganglion tissue at 37 °C. Our findings suggest that mild hypothermic treatment has a beneficial effect on immune system activation after electrode insertion trauma.
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Hipotermia Inducida , Hipotermia , Ratones , Animales , Cóclea , Electrodos Implantados , MacrófagosRESUMEN
BACKGROUND: The air borne SARS-CoV-2 poses a high threat to the elderly and people with underlying diseases. COVID-19 spread quickly in South German nursing homes and for this reason called for preventive measures by the German government. The aim of this paper is to showcase the testing strategies implemented by the Public Health Department Reutlingen to control the spread of COVID-19 in local nursing homes and to report the results thereof. METHODS: This study reports COVID-19 outbreaks in nursing homes in Reutlingen County and how they were dealt with through extensive testing, contact tracing, isolation and hygiene inspections. The testing strategy consisted of three phases: In phase 1 only suspected cases, in phase 2 all staff and residents, and in phase 3 all suspected cases and their contacts were tested. RESULTS: Nearly all residents (98%) and staff members (92%) of all nursing homes in Reutlingen County were tested for SARS-COV-2. 25 of 37 nursing homes had COVID-19 cases, 5 had 30-81 cases/home. 62% of the 395 nursing homes cases were residents, but less than half of them exhibited symptoms (41%). The cases uncovered in nursing homes represented 26% of all 1529 cases in Reutlingen County during the time of this study. CONCLUSIONS: Many COVID-19 cases were discovered through extensive testing, allowing for early interventions. The results shed light on the COVID-19 situation in nursing homes and allowed for individually designed preventive measures. The results also lead to a change in the German legislation. The outbreak management methods of the Public Health Department Reutlingen may also be applicable in other countries.
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COVID-19 , Trazado de Contacto , Anciano , COVID-19/epidemiología , Brotes de Enfermedades , Humanos , Casas de Salud , SARS-CoV-2RESUMEN
BACKGROUND: The analysis of obstructive sleep apnoea syndrome (OSAS) is time- and cost-intensive. A number of studies demonstrated that the non-invasive analysis of exhaled breath (EB) may be suitable to distinguish between OSAS patients and healthy subjects (HS). Methods/Population: We included OSAS patients (n = 15) and HS (n = 15) in this diagnostic proof-of-concept-study. All participants underwent polygraphy to verify or exclude OSAS and performed spirometry to exclude pulmonary ventilatory diseases. The volatile organic compound profile of EB and of the headspaces over EB condensate, pharyngeal washing fluid, and serum was measured using ion mobility spectrometry (IMS) (BioScout®) and an e-nose (Cyranose® 320). For the statistical analysis, we fitted classification tree models using recursive partitioning, followed by a leave-one-out cross-validation. For the cross-validated predictions we calculated descriptive classification statistics, p-values from a [Formula: see text]-test with continuity correction, as well as ROC curves. RESULTS: Using IMS, OSAS patients and HS could be distinguished with high accuracy (values ranged from 79% to 97%). The results of the e-nose-derived analyses (with the exception of EB) were less accurate. However, the cross-validated accuracy for EB was very good (0.9), reflecting a positive predictive value of 100% and a negative predictive value of 83%. For each material, we identified the best five substances that may be used for diagnostic purposes. 2-Methylfluran was found in three different biological materials to be discriminative between OSAS and HS. CONCLUSION: The results strengthen the hypothesis that substances detectable in headspace measurements of different airway and blood materials may undergo a transition from blood into the alveoli (and EB) or vice versa. This means that substances from different compartments could be used to distinguish patients with airway diseases (in this case OSAS) from healthy controls.
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Espectrometría de Movilidad Iónica/métodos , Apnea Obstructiva del Sueño/diagnóstico , Olfato , Pruebas Respiratorias , Nariz Electrónica , Espiración , Femenino , Humanos , Masculino , Persona de Mediana Edad , Alveolos Pulmonares/fisiopatología , Curva ROC , Compuestos Orgánicos Volátiles/análisisRESUMEN
Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K(M) and V(max) values of 23.9(+/-0.96) mM and 192.3 micromol/min mg protein (+/-14.3), respectively, for the native enzyme. For the recombinant counterpart, K(M) was 21.5(+/-0.90) mM and V(max) was 144.9(+/-8.9) micromol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy.
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Arginasa/química , Arginasa/metabolismo , Leishmania/química , Leishmania/enzimología , Animales , Arginasa/genética , Arginina/metabolismo , Escherichia coli/genética , Expresión Génica , Cinética , Microcuerpos/química , Microscopía Fluorescente , Modelos Moleculares , Ornitina/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Urea/metabolismoRESUMEN
Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.
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Interleucinas/química , Receptores de Interleucina/química , Secuencia de Aminoácidos , Reactivos de Enlaces Cruzados/química , Citocinas/química , Citocinas/metabolismo , Dimerización , Electroforesis , Glutaral/química , Humanos , Interleucina-10/química , Interleucina-10/metabolismo , Interleucinas/metabolismo , Datos de Secuencia Molecular , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Receptores de Interleucina/metabolismo , Dispersión de Radiación , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Interleucina-22RESUMEN
Arginase (l-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysisof l-arginine to l-ornithine and urea. In Leishmania spp., the biological role of the enzyme may beinvolved in modulating NO production upon macrophage infection. Previously, we cloned and characterizedthe arginase gene from Leishmania (Leishmania) amazonensis. In the presentwork,we successfullyexpressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterizationof both the native and recombinant enzymes. We obtained KM and Vmax values of 23.9(±0.96)mM and192.3 mol/minmg protein (±14.3), respectively, for the native enzyme. For the recombinant counterpart,KM was 21.5(±0.90)mMand Vmax was 144.9(±8.9) mol/min mg. Antibody against the recombinantprotein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data fromlight scatteringand small angle X-ray scattering showed that a trimeric state is the active form of the protein.Wedetermined empirically that a manganesewash at room temperature is the best condition to purify activeenzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirmthe structural disposition of histidine at positions 3 and 324. The determined structural parametersprovide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase,which could subsequently point to a candidate for leishmaniasis therapy.
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Masculino , Femenino , Humanos , Leishmania/genética , Leishmania/inmunología , Leishmania/metabolismo , ArginasaRESUMEN
The thyroid hormone receptor (TR) D-domain links the ligand-binding domain (LBD, EF-domain) to the DNA-binding domain (DBD, C-domain), but its structure, and even its existence as a functional unit, are controversial. The D domain is poorly conserved throughout the nuclear receptor family and was originally proposed to comprise an unfolded hinge that facilitates rotation between the LBD and the DBD. Previous TR LBD structures, however, have indicated that the true unstructured region is three to six amino acid residues long and that the D-domain N terminus folds into a short amphipathic alpha-helix (H0) contiguous with the DBD and that the C terminus of the D-domain comprises H1 and H2 of the LBD. Here, we solve structures of TR-LBDs in different crystal forms and show that the N terminus of the TRalpha D-domain can adopt two structures; it can either fold into an amphipathic helix that resembles TRbeta H0 or form an unstructured loop. H0 formation requires contacts with the AF-2 coactivator-binding groove of the neighboring TR LBD, which binds H0 sequences that resemble coactivator LXXLL motifs. Structural analysis of a liganded TR LBD with small angle X-ray scattering (SAXS) suggests that AF-2/H0 interactions mediate dimerization of this protein in solution. We propose that the TR D-domain has the potential to form functionally important extensions of the DBD and LBD or unfold to permit TRs to adapt to different DNA response elements. We also show that mutations of the D domain LXXLL-like motif indeed selectively inhibit TR interactions with an inverted palindromic response element (F2) in vitro and TR activity at this response element in cell-based transfection experiments.
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Receptores alfa de Hormona Tiroidea/química , Receptores alfa de Hormona Tiroidea/metabolismo , Receptores beta de Hormona Tiroidea/química , Receptores beta de Hormona Tiroidea/metabolismo , Secuencias de Aminoácidos , ADN/metabolismo , Dimerización , Células HeLa , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Elementos de Respuesta/genética , Soluciones , Relación Estructura-Actividad , Triyodotironina/metabolismo , Células Tumorales Cultivadas , Difracción de Rayos XRESUMEN
We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.
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Amidohidrolasas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Thermotoga maritima/enzimología , Zinc/químicaRESUMEN
The enzyme beta-xylosidase from Trichoderma reesei, a member of glycosil hydrolase family 3 (GH3), is a glycoside hydrolase which acts at the glycosidic linkages of 1,4-beta-xylooligosaccharides and that also exhibits alpha-l-arabinofuranosidase activity on 4-nitrophenyl alpha-l-arabinofuranoside. In this work, we show that the enzyme forms monomers in solution and derive the low-resolution molecular envelope of the beta-xylosidase from small-angle X-ray scattering (SAXS) data using the ab initio simulated annealing algorithm. The radius of gyration and the maximum dimension of the beta-xylosidase are 30.3 +/- 0.2 and 90 +/- 5 A, respectively. In contrast to the fold of the only two structurally characterized members of GH3, the barley beta-d-glucan exohydrolase and beta-hexosaminidase from Vibrio cholerae, which have respectively two or one distinct domains, the shape of the beta-xylosidase indicates the presence of three distinct structural modules. Domain recognition algorithms were used to show that the C-terminal part of the amino acid sequence of the protein forms the third domain. Circular dichroism spectroscopy and secondary structure prediction programs demonstrate that this additional domain adopts a predominantly beta conformation.
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Trichoderma/enzimología , Xilosidasas/química , Algoritmos , Dicroismo Circular , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Dispersión de Radiación , Soluciones , Rayos XRESUMEN
OBJECTIVES: A rational approach in the treatment of chronic rhinosinusitis (CRS) is the intranasal application of antiseptic agents, due to the pathogenetic role of bacteria and fungi. N-Chlorotaurine (NCT), a mild endogenous oxidant with broad-spectrum antimicrobial activity, has been tested for the first time in CRS. METHODS: This one-arm phase IIa clinical study is the first step in the clinical development of this promising substance for local therapy of CRS. The nasal and paranasal cavities of 12 patients were rinsed with 10-20 ml of 1% aqueous NCT solution, applied via a novel catheter system (YAMIK). Treatment consisted of three lavages per week for 4 weeks. RESULTS: NCT caused neither alterations of the mucosa nor burning pain during application. Nevertheless, the insertion of the catheter, the insufflation of the posterior cuff and the overpressure inside the sinuses after infiltration led to moderate pain in some patients. Mucosal swelling decreased in all subjects, nasal breathing could be improved in nine patients and impaired olfaction in seven. Polyps did not disappear within the 1-month period of the study. CONCLUSIONS: The good tolerability and possible beneficial effects of NCT encourage its further investigation in CRS. Despite some limitations the YAMIK catheter proved to be a convenient and safe device for rinsing the nasal and paranasal sinuses.
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Antiinfecciosos/administración & dosificación , Sinusitis/tratamiento farmacológico , Taurina/análogos & derivados , Adulto , Cateterismo/instrumentación , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Espectrofotometría , Taurina/administración & dosificación , Irrigación TerapéuticaRESUMEN
Proteins that belong to the heat shock protein (Hsp) 40 family assist Hsp70 in many cellular functions and are important for maintaining cell viability. A knowledge of the structural and functional characteristics of the Hsp40 family is therefore essential for understanding the role of the Hsp70 chaperone system in cells. In this work, we used small angle x-ray scattering and analytical ultracentrifugation to study two representatives of human Hsp40, namely, DjA1 (Hdj2/dj2/HSDJ/Rdj1) from subfamily A and DjB4 (Hlj1/DnaJW) from subfamily B, and to determine their quaternary structure. We also constructed low resolution models for the structure of DjA1-(1-332), a C-terminal-deleted mutant of DjA1 in which dimer formation is prevented. Our results, together with the current structural information of the Hsp40 C-terminal and J-domains, were used to generate models of the internal structural organization of DjA1 and DjB4. The characteristics of these models indicated that DjA1 and DjB4 were both dimers, but with substantial differences in their quaternary structures: whereas DjA1 consisted of a compact dimer in which the N and C termini of the two monomers faced each other, DjB4 formed a dimer in which only the C termini of the two monomers were in contact. The two proteins also differed in their ability to bind unfolded luciferase. Overall, our results indicate that these representatives of subfamilies A and B of human Hsp40 have different quaternary structures and chaperone functions.
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Proteínas de Choque Térmico/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/aislamiento & purificación , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Datos de Secuencia Molecular , Pliegue de Proteína , Alineación de Secuencia , SolucionesRESUMEN
The mass density of proteins is a relevant basic biophysical quantity. It is also a useful input parameter, for example, for three-dimensional structure determination by protein crystallography and studies of protein oligomers in solution by analytic ultracentrifugation. We have performed a critical analysis of published, theoretical, and experimental investigations about this issue and concluded that the average density of proteins is not a constant as often assumed. For proteins with a molecular weight below 20 kDa, the average density exhibits a positive deviation that increases for decreasing molecular weight. A simple molecular-weight-depending function is proposed that provides a more accurate estimate of the average protein density.
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Proteínas/química , Biología Computacional/métodos , Cristalografía , Peso Molecular , Conformación Proteica , UltracentrifugaciónRESUMEN
The alpha zein, the maize storage prolamin, is a mixture of several homologous polypeptides that shows two bands in SDS-PAGE, called Z19 and Z22. The conformation studies carried out by several authors in this mixture are conflicting. To elucidate these inconsistencies, we analyzed the conformation of the Z19 fraction, extracted from BR451 maize variety by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and small-angle X-ray scattering. The infrared results show that Z19 has 46% of alpha helix and 22% of beta sheet. The fast N-H to N-D exchange measured by (1)H NMR spectroscopy showed that Z19 is not a compact structure. The scattering measurements indicated an extended structure with 12 by 130 A. With these data, we have modeled the Z19 structure as a hairpin, composed of helical, sheet, turns, and secondary structures, folded back on itself.
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Conformación Proteica , Zea mays/química , Zeína/química , Espectroscopía de Resonancia Magnética , Dispersión de Radiación , Espectroscopía Infrarroja por Transformada de Fourier , Rayos XRESUMEN
Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize ( Zea mays) and pearl millet ( Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high alpha-helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, (1)H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded alpha-helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.
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Modelos Moleculares , Pennisetum/metabolismo , Proteínas de Plantas/química , Análisis Espectral/métodos , Zea mays/metabolismo , Alcoholes/química , Simulación por Computador , Etanol/química , Prolaminas , Conformación Proteica , SolucionesRESUMEN
The co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK.
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Proteínas de Choque Térmico/química , Mitocondrias/química , Chaperonas Moleculares , Secuencia de Aminoácidos , Secuencia de Bases , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Clonación Molecular , Simulación por Computador , Cartilla de ADN , Dimerización , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Soluciones , Termodinámica , Difracción de Rayos XRESUMEN
The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme. When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure. Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level. We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures. The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex. Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer. We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure. Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure. Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.
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Escherichia coli/enzimología , Fosfofructoquinasa-2/química , Estructura Cuaternaria de Proteína , Ligandos , Modelos Moleculares , Fosfofructoquinasa-2/aislamiento & purificación , Fosfofructoquinasa-2/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Soluciones , Difracción de Rayos XRESUMEN
Nuclear receptors are ligand-inducible transcription factors that share structurally related DNA-binding (DBD) and ligand-binding (LBD) domains. Biochemical and structural studies have revealed the modular nature of DBD and LBD. Nevertheless, the domains function in concert in vivo. While high-resolution crystal structures of nuclear receptor DBDs and LBDs are available, there are no x-ray structural studies of nuclear receptor proteins containing multiple domains. We report the solution structures of the human retinoid X receptor DBD-LBD (hRXRalphaDeltaAB) region. We obtained ab initio shapes of hRXRalphaDeltaAB dimer and tetramer to 3.3 and 1.7 nm resolutions, respectively, and established the position and orientation of the DBD and LBD by fitting atomic coordinates of hRXRalpha DBD and LBD. The dimer is U-shaped with DBDs spaced at approximately 2 nm in a head to head orientation forming an angle of about 10 degrees with respect to each other and with an extensive interface area provided by the LBD. The tetramer is a more elongated X-shaped molecule formed by two dimers in head to head arrangement in which the DBDs are extended from the structure and spaced at about 6 nm. The close proximity of DBDs in dimers may facilitate homodimer formation on DNA; however, for the homodimer to bind to a DNA element containing two directly repeated half-sites, one of the DBDs would need to rotate with respect to the other element. By contrast, the separation of DBDs in the tetramers may account for their decreased ability to recognize DNA.
Asunto(s)
ADN/metabolismo , Receptores de Ácido Retinoico/química , Factores de Transcripción/química , Sitios de Unión , Dimerización , Ligandos , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Dispersión de Radiación , Soluciones , Factores de Transcripción/metabolismo , Rayos XRESUMEN
Classical parameters obtained from surface tension technique coupled to small angle X-ray scattering (SAXS) measurements gave support to investigate conformational changes in the bovine serum albumin (BSA)-sodium dodecyl sulfate (SDS) complexes, as well as the size of the micelle-like clusters distributed along the polypeptide chain. The studied systems were composed of 1 wt% of BSA in the absence and presence of increasing SDS molar concentration up to 80 mM, under experimental conditions of low ionic strength and pH 5.40. At SDS concentrations below the critical aggregation concentration (cac) of 2.2 mM, SAXS results indicate that the detergent does not modify the native protein conformation. However, the beginning of protein unfolding, evidenced by SAXS through an increase in the values of radius of gyration Rg and protein maximum dimension Dmax, is coincident with the onset of SDS cooperative binding to BSA identified by the first breakpoint in the surface tension-SDS profile. Further SDS addition leads to the formation of micelle-like aggregates randomly distributed along the unfolded polypeptide chain, consistent to a necklace and bead model. The SAXS data also demonstrate that the SDS micelles grow in size up to 50 mM detergent. At 50 mM surfactant, the micelles stop growing. This concentration is near the BSA saturation binding by SDS measured by dialyzes and indicated by the second breakpoint in surface tension-SDS profile. The SAXS and surface tension data are also consistent with the formation of free micelles in equilibrium with BSA-SDS complexes for surfactant amount above the saturation.