RESUMEN
Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, "S-XL6," was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A's in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Animales , Ratones , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Cisteína/genética , Mutación , Superóxido Dismutasa/genética , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genéticaRESUMEN
Human ornithine transcarbamylase (hOTC) is a mitochondrial transferase protein involved in the urea cycle and is crucial for the conversion of toxic ammonia to urea. Structural analysis coupled with kinetic studies of Escherichia coli, rat, bovine, and other transferase proteins has identified residues that play key roles in substrate recognition and conformational changes but has not provided direct evidence for all of the active residues involved in OTC function. Here, computational methods were used to predict the likely active residues of hOTC; the function of these residues was then probed with site-directed mutagenesis and biochemical characterization. This process identified previously reported active residues, as well as distal residues that contribute to activity. Mutation of active site residue D263 resulted in a substantial loss of activity without a decrease in protein stability, suggesting a key catalytic role for this residue. Mutation of predicted second-layer residues H302, K307, and E310 resulted in significant decreases in enzymatic activity relative to that of wild-type (WT) hOTC with respect to l-ornithine. The mutation of fourth-layer residue H107 to produce the hOTC H107N variant resulted in a 66-fold decrease in catalytic efficiency relative to that of WT hOTC with respect to carbamoyl phosphate and a substantial loss of thermal stability. Further investigation identified H107 and to a lesser extent E98Q as key residues involved in maintaining the hOTC quaternary structure. This work biochemically demonstrates the importance of D263 in hOTC catalytic activity and shows that residues remote from the active site also play key roles in activity.
Asunto(s)
Dominio Catalítico , Mutagénesis Sitio-Dirigida , Ornitina Carbamoiltransferasa , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Ornitina Carbamoiltransferasa/química , Humanos , Modelos Moleculares , Cinética , Estabilidad de Enzimas , CatálisisRESUMEN
Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas Proto-Oncogénicas p21(ras)/química , Quinasas raf/química , Galectinas/química , Galectinas/genética , Galectinas/metabolismo , Humanos , Dominios Proteicos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
HRas, KRas, and NRas are GTPases with a common set of effectors that control many cell-signaling pathways, including proliferation through Raf kinase. Their G-domains are nearly identical in sequence, with a few isoform-specific residues that have an effect on dynamics and biochemical properties. Here, we use accelerated molecular dynamics (aMD) simulations consistent with solution x-ray scattering experiments to elucidate mechanisms through which isoform-specific residues associated with each Ras isoform affects functionally important regions connected to the active site. HRas-specific residues cluster in loop 8 to stabilize the nucleotide-binding pocket, while NRas-specific residues on helix 3 directly affect the conformations of switch I and switch II. KRas, the most globally flexible of the isoforms, shows greatest fluctuations in the switch regions enhanced by a KRas-specific residue in loop 7 and a highly dynamic loop 8 region. The analysis of isoform-specific residue effects on Ras proteins is supported by NMR experiments and is consistent with previously published biochemical data.
Asunto(s)
Nucleótidos , Proteínas ras , Guanosina Trifosfato/metabolismo , Mutación , Nucleótidos/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinasas raf/metabolismo , Proteínas ras/metabolismoRESUMEN
NF-κB essential modulator (NEMO) regulates NF-κB signaling by acting as a scaffold for the kinase IKKß to direct its activity toward the NF-κB inhibitor, IκBα. Here, we show that a highly conserved central region of NEMO termed the intervening domain (IVD, amino acids 112-195) plays a key role in NEMO function. We determined a structural model of full-length NEMO by small-angle X-ray scattering and show that full-length, wild-type NEMO becomes more compact upon binding of a peptide comprising the NEMO binding domain of IKKß (amino acids 701-745). Mutation of conserved IVD residues (9SG-NEMO) disrupts this conformational change in NEMO and abolishes the ability of NEMO to propagate NF-κB signaling in cells, although the affinity of 9SG-NEMO for IKKß compared to that of the wild type is unchanged. On the basis of these results, we propose a model in which the IVD is required for a conformational change in NEMO that is necessary for its ability to direct phosphorylation of IκBα by IKKß. Our findings suggest a molecular explanation for certain disease-associated mutations within the IVD and provide insight into the role of conformational change in signaling scaffold proteins.
Asunto(s)
Quinasa I-kappa B/metabolismo , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Quinasa I-kappa B/química , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Transducción de Señal , Difracción de Rayos XRESUMEN
Adenylate kinase (ADK) catalyzes the reversible Mg2+ -dependent phosphoryl transfer reaction Mg2+ +2ADP âMg2+ +ATP + AMP in essential cellular systems. This reaction is a major player in cellular energy homeostasis and the isoform network of ADK plays an important role in AMP metabolic signaling circuits. ADK has 3 domains, the LID, NMP, and CORE domains, that undergo large conformational rearrangements during ADK's catalytic cycle. In spite of extensive experimental and computational studies, details of the conformational pathway from open to closed forms remain uncertain. In this paper we explore this pathway using coarse-grained molecular dynamics (MD) trajectories of ADK calculated by GROMACS using a SMOG model and classify the conformations within the resultant trajectories by K-means clustering. ADK conformations segregate naturally into open; intermediate; and closed forms with long-term residence in the intermediate state. Structural clustering divides the intermediate conformation into 3 sub-states that are distinguished from one another on the basis of differences in both structure and dynamics. These distinctions are defined on the basis of a number of different metrics including radius of gyration, dihedral angle fluctuation, and fluctuations of interatomic pair distances. Furthermore, differences in the sub-states appear to correspond to the distinct ways each sub-state contributes to the molecular mechanism of catalysis: One sub-state acts as a gate-way to the open conformation; one sub-state a gate-way to the closed conformation. A third intermediate sub-state appears to represent a metastable off-pathway structure that is nevertheless frequently visited during the passage from open to closed state.
Asunto(s)
Adenilato Quinasa/química , Proteínas de Escherichia coli/química , Simulación de Dinámica Molecular , Conformación Proteica , Adenilato Quinasa/metabolismo , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Proteínas de Escherichia coli/metabolismo , CinéticaRESUMEN
The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The P-CR structure was fit into a molecular envelope for the P-CR domain derived from small-angle X-ray scattering data. The P-CR structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecular envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the P-CR domain from the molecular docking models places the P-CR connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the P-CR coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery.
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Glucosiltransferasas/química , Oryza/química , Proteínas de Plantas/química , Dominio Catalítico , Cristalografía por Rayos X , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformación Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Crystal structures of adenylate kinase (AdK) from Escherichia coli capture two states: an "open" conformation (apo) obtained in the absence of ligands and a "closed" conformation in which ligands are bound. Other AdK crystal structures suggest intermediate conformations that may lie on the transition pathway between these two states. To characterize the transition from open to closed states in solution, X-ray solution scattering data were collected from AdK in the apo form and with progressively increasing concentrations of five different ligands. Scattering data from apo AdK are consistent with scattering predicted from the crystal structure of AdK in the open conformation. In contrast, data from AdK samples saturated with Ap5A do not agree with that calculated from AdK in the closed conformation. Using cluster analysis of available structures, we selected representative structures in five conformational states: open, partially open, intermediate, partially closed, and closed. We used these structures to estimate the relative abundances of these states for each experimental condition. X-ray solution scattering data obtained from AdK with AMP are dominated by scattering from AdK in the open conformation. For AdK in the presence of high concentrations of ATP and ADP, the conformational ensemble shifts to a mixture of partially open and closed states. Even when AdK is saturated with Ap5A, a significant proportion of AdK remains in a partially open conformation. These results are consistent with an induced-fit model in which the transition of AdK from an open state to a closed state is initiated by ATP binding.
Asunto(s)
Adenosina Difosfato/química , Adenosina Trifosfato/química , Adenilato Quinasa/química , Fosfatos de Dinucleósidos/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Adenilato Quinasa/genética , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli/genéticaRESUMEN
An alternate formulation of helical diffraction theory is used to generate cross-sectional shapes of fibrous structures from equatorial scattering. We demonstrate this approach with computationally generated scattering intensities and then apply it to scattering data from Tobacco Mosaic Virus (TMV) and in vitro assembled fibrils of Aß40 peptides. Refining the cross-sectional shape of TMV from SAXS data collected on a 26mg/ml solution resulted in a circular shape with outer diameter of â¼180Å and inner diameter of â¼40Å consistent with the known structure of TMV. We also utilized this method to analyze the equatorial scattering from TMV collected by Don Caspar from a concentrated (24% â¼295mg/ml) gel of TMV as reported in his Ph.D. thesis in 1955. This data differs from the SAXS data in having a sharp interference peak at â¼250Å spacing, indicative of strong interparticle interactions in the gel. Analysis of this data required consideration of interatomic vectors as long as 2000Å and resulted in generation of images that were interpreted as representative of local organization of TMV particles in the sample. Peaks in the images were separated, on average by about 250Å with a density consistent with Caspar's original measurements. Analysis of SAXS data from Aß fibrils resulted in a cross-sectional shape that could be interpreted in terms of structural models that have been constructed from ssNMR and cryoEM. These results demonstrate an unexpected use of the small-angle region of fiber diffraction patterns to derive fundamental structural properties of scattering objects.
Asunto(s)
Péptidos beta-Amiloides/química , Modelos Teóricos , Virus del Mosaico del Tabaco/química , Amiloide/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Cellulose microfibrils are para-crystalline arrays of several dozen linear (1â4)-ß-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.
Asunto(s)
Dominio Catalítico , Glucosiltransferasas/química , Oryza/enzimología , Membrana Celular/metabolismo , Pared Celular/metabolismo , Celulosa/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Modelos Moleculares , Conformación Molecular , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes , Especificidad por SustratoRESUMEN
There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.
Asunto(s)
Proteínas de Neoplasias/sangre , Neoplasias/metabolismo , Péptidos/análisis , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Marcaje Isotópico , Espectrometría de Masas/métodos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/aislamiento & purificación , Neoplasias/sangre , Péptidos/química , Reproducibilidad de los ResultadosRESUMEN
Extending collection of x-ray solution scattering data into the wide-angle regime (WAXS) can provide information not readily extracted from small angle (SAXS) data. It is possible to accurately predict WAXS scattering on the basis of atomic coordinate sets and thus use it as a means of testing molecular models constructed on the basis of crystallography, molecular dynamics (MD), cryo-electron microscopy or ab initio modeling. WAXS data may provide insights into the secondary, tertiary and quaternary structural organization of macromolecules. It can provide information on protein folding and unfolding beyond that attainable from SAXS data. It is particularly sensitive to structural fluctuations in macromolecules and can be used to generate information about the conformational make up of ensembles of structures co-existing in solution. Novel approaches to modeling of structural fluctuations can provide information on the spatial extent of large-scale structural fluctuations that are difficult to obtain by other means. Direct comparison with the results of MD simulations are becoming possible. Because it is particularly sensitive to small changes in structure and flexibility it provides unique capabilities for the screening of ligand libraries for detection of functional interactions. WAXS thereby provides an important extension of SAXS that can generate structural and dynamic information complementary to that obtainable by other biophysical techniques.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas/ultraestructura , Difracción de Rayos X/métodos , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Humanos , Difracción de Neutrones/instrumentación , Difracción de Neutrones/métodos , Conformación Proteica , Pliegue de Proteína , Proteínas/química , Dispersión del Ángulo Pequeño , Homología Estructural de Proteína , Difracción de Rayos X/instrumentaciónRESUMEN
Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity.
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Ácido Mirístico/metabolismo , Proteínas Proto-Oncogénicas c-abl/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación/genética , Conformación Proteica , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Transducción de Señal , Soluciones , Resonancia por Plasmón de Superficie , Dominios Homologos srcRESUMEN
Molecular dynamics (MD) trajectories are very large data sets that contain substantial information about the dynamic behavior of a protein. Condensing these data into a form that can provide intuitively useful understanding of the molecular behavior during the trajectory is a substantial challenge that has received relatively little attention. Here, we introduce the sigma-r plot, a plot of the standard deviation of intermolecular distances as a function of that distance. This representation of global dynamics contains within a single, one-dimensional plot, the average range of motion between pairs of atoms within a macromolecule. Comparison of sigma-r plots calculated from 10 ns trajectories of proteins representing the four major SCOP fold classes indicates diversity of dynamic behaviors which are recognizably different among the four classes. Differences in domain structure and molecular weight also produce recognizable features in sigma-r plots, reflective of differences in global dynamics. Plots generated from trajectories with progressively increasing simulation time reflect the increased sampling of the structural ensemble as a function of time. Single amino acid replacements can give rise to changes in global dynamics detectable through comparison of sigma-r plots. Dynamic behavior of substructures can be monitored by careful choice of interatomic vectors included in the calculation. These examples provide demonstrations of the utility of the sigma-r plot to provide a simple measure of the global dynamics of a macromolecule.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas/química , Animales , Bases de Datos de Proteínas , Infecciones por VIH/virología , Proteasa del VIH/química , VIH-1/química , VIH-1/enzimología , Humanos , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion-limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM-IBP fusion polypeptides composed of a carbohydrate-binding module family 11 (CBM11) and an iron-binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM-IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM-IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM-IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.
Asunto(s)
Arabidopsis/metabolismo , Biomasa , Pared Celular/metabolismo , Hierro/metabolismo , Oryza/metabolismo , Semillas/metabolismo , Arabidopsis/genética , Biocombustibles , Pared Celular/genética , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
The flexibility of HIV protease (HIVp) plays a critical role in enabling enzymatic activity and is required for substrate access to the active site. While the importance of flexibility in the flaps that cover the active site is well known, flexibility in other parts of the enzyme is also critical for function. One key region is a loop containing Thr 80, which forms the walls of the active site. Although not situated within the active site, amino acid Thr80 is absolutely conserved. The mutation T80N preserves the structure of the enzyme but catalytic activity is completely lost. To investigate the potential influence of the T80N mutation on HIVp flexibility, wide-angle X-ray scattering (WAXS) data was measured for a series of HIVp variants. Starting with a calculated WAXS pattern from a rigid atomic model, the modulations in the intensity distribution caused by structural fluctuations in the protein were predicted by simple analytic methods and compared with the experimental data. An analysis of T80N WAXS data shows that this variant is significantly more rigid than the WT across all length scales. The effects of this single point mutation extend throughout the protein, to alter the mobility of amino acids in the enzymatic core. These results support the contentions that significant protein flexibility extends throughout HIVp and is critical to catalytic function.
Asunto(s)
Proteasa del VIH/química , Proteasa del VIH/metabolismo , Proteasa del VIH/genética , Simulación de Dinámica Molecular , Mutación , Docilidad , Difracción de Rayos XRESUMEN
Cellulose is an attractive candidate as a feedstock for sustainable bioenergy because of its global abundance. Pretreatment of biomass has significant influence on the chemical availability of cellulose locked in recalcitrant microfibrils. Optimizing pretreatment depends on an understanding of its impact on the microscale and nanoscale molecular architecture. X-ray scattering experiments have been performed on native and pre-treated maize stover and models of cellulose architecture have been derived from these data. Ultra small-angle, very small-angle and small-angle X-ray scattering (USAXS, VSAXS and SAXS) probe three different levels of architectural scale. USAXS and SAXS have been used to study cellulose at two distinct length scales, modeling the fibrils as ~30 Å diameter rods packed into ~0.14 µm diameter bundles. VSAXS is sensitive to structural features at length scales between these two extremes. Detailed analysis of diffraction patterns from untreated and pretreated maize using cylindrical Guinier plots and the derivatives of these plots reveals the presence of substructures within the ~0.14 µm diameter bundles that correspond to grouping of cellulose approximately 30 nm in diameter. These sub-structures are resilient to dilute acid pretreatments but are sensitive to pretreatment when iron sulfate is added. These results provide evidence of the hierarchical arrangement of cellulose at three length scales and the evolution of these arrangements during pre-treatments.
RESUMEN
In this paper, we describe a model for maximum likelihood estimation (MLE) of the relative abundances of different conformations of a protein in a heterogeneous mixture from small angle X-ray scattering (SAXS) intensities. To consider cases where the solution includes intermediate or unknown conformations, we develop a subset selection method based on k-means clustering and the Cramér-Rao bound on the mixture coefficient estimation error to find a sparse basis set that represents the space spanned by the measured SAXS intensities of the known conformations of a protein. Then, using the selected basis set and the assumptions on the model for the intensity measurements, we show that the MLE model can be expressed as a constrained convex optimization problem. Employing the adenylate kinase (ADK) protein and its known conformations as an example, and using Monte Carlo simulations, we demonstrate the performance of the proposed estimation scheme. Here, although we use 45 crystallographically determined experimental structures and we could generate many more using, for instance, molecular dynamics calculations, the clustering technique indicates that the data cannot support the determination of relative abundances for more than 5 conformations. The estimation of this maximum number of conformations is intrinsic to the methodology we have used here.
RESUMEN
A new way to use wide-angle x-ray solution scattering to study protein-ligand binding is presented. First, scattering patterns are measured at different protein and ligand concentrations. Multivariate curve resolution based on singular value decomposition and global analysis is applied to estimate the binding affinities and reference patterns (i.e., the scattering patterns of individual components). As validated by simulation, Bayesian confidence intervals provide accurate uncertainty estimates for the binding free energies and reference patterns. Experimental results from several protein-ligand systems demonstrate the feasibility of the approach, which promises to expand the role of wide-angle x-ray scattering as a quantitative biophysical tool.
Asunto(s)
Proteínas/química , Difracción de Rayos X , Acetilglucosamina/metabolismo , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/metabolismo , Animales , Teorema de Bayes , Pollos , Galectinas/química , Galectinas/metabolismo , Humanos , Lactosa/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Análisis Multivariante , Muramidasa/química , Muramidasa/metabolismo , Niacinamida/metabolismo , Unión ProteicaRESUMEN
The Arabidopsis stem is composed of five tissues - the pith, xylem, phloem, cortex and epidermis - each of which fulfills specific roles in support of the growth and survival of the organism. The lignocellulosic scaffolding of cell walls is specialized to provide optimal support for the diverse functional roles of these layers, but little is known about this specialization. X-ray scattering can be used to study this tissue-specific diversity because the cellulosic components of the cell walls give rise to recognizable scattering features interpretable in terms of the underlying molecular architecture and distinct from the largely unoriented scatter from other constituents. Here we use scanning X-ray microdiffraction from thin sections to characterize the diversity of molecular architecture in the Arabidopsis stem and correlate that diversity to the functional roles the distinct tissues of the stem play in the growth and survival of the organism.