4,688 research outputs found

    Analysis Of Slit-Distorted Small-Angle X-Ray Scattering Intensities Without Desmearing

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    Experimental small-angle X-ray scattering intensities, generated from a primary beam of known intensity profile, are often \u27desmeared\u27 to obtain point-collimated intensities. A much simpler way is shown of using the known beam intensity profile to derive, from the experimental scattering intensity, the quantities required for calculation of surface areas

    Small-Angle X-Ray Scattering Analysis Of Catalysts: Comparison and Evaluation Of Models

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    Small-angle X-ray scattering (SAXS) can be used to obtain interphase surface areas of a system, such as a supported-metal catalyst, composed of internally homogeneous phases with sharp interphase boundaries. Measurements of SAXS for samples of porous silica, alumina, platinum on silica, and platinum on alumina are reported. A variety of models and forms for the correlation function, the Fourier transform of which gives the X-ray scattering, are considered, and theoretical and measured intensities are compared. A criterion of fit for comparing models with different numbers of parameters is proposed. It is shown that values for the single interphase surface area can be obtained independently of a model. However, fitting intensities using a model-based correlation function gives information about the structure of the system. The two-cell-size Voronoi and the correlated Voronoi cell models are useful in this regard

    FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR)

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    Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I)

    Active-Site-Directed Inhibitors of Prolyl Oligopeptidase Abolish Its Conformational Dynamics

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    Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond–millisecond motions opens the opportunity for regulating protein–protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation.This work was supported by the Institute for Research in Biomedicine, MINECO-FEDER (Bio2013-40716-R, CTQ2013-48287 and CTQ2012-32183/BQU), and the Generalitat de Catalunya (XRB and Grup Consolidat 2014SGR521). AL has received funding from the Instituto de Salud Carlos III. PB acknowledges the Agence Nationale de la Recherche (SPINHD-ANR-CHEX-2011) and the ATIP-Avenir program for financial support. FHT’s fellowship is co-funded by the INSERM and the University of Copenhagen. Technical assistance from staff at the P12 beam line (EMBL/DESY) is acknowledged.Peer ReviewedPostprint (author's final draft

    De novo design of a homo-trimeric amantadine-binding protein.

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    The computational design of a symmetric protein homo-oligomer that binds a symmetry-matched small molecule larger than a metal ion has not yet been achieved. We used de novo protein design to create a homo-trimeric protein that binds the C3 symmetric small molecule drug amantadine with each protein monomer making identical interactions with each face of the small molecule. Solution NMR data show that the protein has regular three-fold symmetry and undergoes localized structural changes upon ligand binding. A high-resolution X-ray structure reveals a close overall match to the design model with the exception of water molecules in the amantadine binding site not included in the Rosetta design calculations, and a neutron structure provides experimental validation of the computationally designed hydrogen-bond networks. Exploration of approaches to generate a small molecule inducible homo-trimerization system based on the design highlight challenges that must be overcome to computationally design such systems

    MetaboLab - advanced NMR data processing and analysis for metabolomics

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    Background\ud Despite wide-spread use of Nuclear Magnetic Resonance (NMR) in metabolomics for the analysis of biological samples there is a lack of graphically driven, publicly available software to process large one and two-dimensional NMR data sets for statistical analysis.\ud \ud Results\ud Here we present MetaboLab, a MATLAB based software package that facilitates NMR data processing by providing automated algorithms for processing series of spectra in a reproducible fashion. A graphical user interface provides easy access to all steps of data processing via a script builder to generate MATLAB scripts, providing an option to alter code manually. The analysis of two-dimensional spectra (1H,13C-HSQC spectra) is facilitated by the use of a spectral library derived from publicly available databases which can be extended readily. The software allows to display specific metabolites in small regions of interest where signals can be picked. To facilitate the analysis of series of two-dimensional spectra, different spectra can be overlaid and assignments can be transferred between spectra. The software includes mechanisms to account for overlapping signals by highlighting neighboring and ambiguous assignments.\ud \ud Conclusions\ud The MetaboLab software is an integrated software package for NMR data processing and analysis, closely linked to the previously developed NMRLab software. It includes tools for batch processing and gives access to a wealth of algorithms available in the MATLAB framework. Algorithms within MetaboLab help to optimize the flow of metabolomics data preparation for statistical analysis. The combination of an intuitive graphical user interface along with advanced data processing algorithms facilitates the use of MetaboLab in a broader metabolomics context.\ud \u

    Crystal structure of monomeric human β-2- microglobulin reveals clues to its amyloidogenic properties

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    Dissociation of human β-2-microglobulin (β(2)m) from the heavy chain of the class I HLA complex is a critical first step in the formation of amyloid fibrils from this protein. As a consequence of renal failure, the concentration of circulating monomeric β(2)m increases, ultimately leading to deposition of the protein into amyloid fibrils and development of the disorder, dialysis-related amyloidosis. Here we present the crystal structure of a monomeric form of human β(2)m determined at 1.8-Å resolution that reveals remarkable structural changes relative to the HLA-bound protein. These involve the restructuring of a β bulge that separates two short β strands to form a new six-residue β strand at one edge of this β sandwich protein. These structural changes remove key features proposed to have evolved to protect β sheet proteins from aggregation [Richardson, J.&Richardson, D. (2002) Proc. Natl. Acad. Sci. USA 99, 2754–2759] and replaces them with an aggregationcompetent surface. In combination with solution studies using (1)H NMR, we show that the crystal structure presented here represents a rare species in solution that could provide important clues about the mechanism of amyloid formation from the normally highly soluble native protein

    Solution structure of the inhibitory phosphorylation domain of myosin phosphatase targeting subunit 1.

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    Cell motility, such as smooth muscle contraction and cell migration, is controlled by the reversible phosphorylation of the regulatory light chain of myosin II and other cytoskeletal proteins. Mounting evidence suggests that in smooth muscle cells and other types of cells in vertebrates, myosin phosphatase (MP) plays an important role in controlling the phosphorylation of myosin II as well as other cytoskeletal proteins, including ezrin, moesin, and radixin.1 MP is a holoenzyme consisting of a catalytic subunit of a type-1 Ser/Thr phosphatase (PP1C) delta isoform, a myosin phosphatase targeting subunit 1 (MYPT1), and an accessory subunit M21. In this ternary complex, MYPT1 is responsible for regulating the phosphatase activity.1 A recent X-ray crystallographic study revealed an allosteric interaction between PP1C and the N-terminal ankyrin repeat domain of MYPT1 that confers the substrate specificity of the enzyme.2 MP activity is suppressed when Thr696 or Thr853 of MYPT1 is phosphorylated by various kinases, such as ROCK, ZIPK, ILK, and PAK.1,3 However, it is still unclear how the phosphorylation of MYPT1 inhibits MP activity. The amino acid sequence around Thr696 of MYPT1 is highly conserved among MYPT1 family members including MYPT2 and MBS85. Therefore, structural insights into the inhibitory domain of MYPT1 are expected to provide new clues to fully elucidate the mechanism that controls phosphatase activity via the phosphorylation of MYPT1 or other family members involved in kinase-phosphatase crosstalk in cytoskeletal regulation. Here, we prepared a bacterial recombinant fragment of MYPT1 corresponding to residues 658 to 714, including the phosphorylation site Thr696, and determined its three-dimensional structure through the use of computer-assisted distance geometry and a simulated annealing protocol combined with stable-isotope-aided multi-dimensional NMR techniques

    A single aromatic residue in transcriptional repressor protein KorA is critical for cooperativity with its co-regulator KorB

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    A central feature of broad host range IncP-1 plasmids is the set of regulatory circuits that tightly control plasmid core functions under steady-state conditions. Cooperativity between KorB and either KorA or TrbA repressor proteins is a key element of these circuits and deletion analysis has implicated the conserved C-terminal domain of KorAand TrbAin this interaction. By NMR we show that KorA and KorB interact directly and identify KorA amino acids that are affected on KorB binding. Studies on mutants showed that tyrosine 84 (or phenylalanine, in some alleles) is dispensable for repressor activity but critical for the specific interaction with KorB in both in vivo reporter gene assays and in vitro electrophoretic mobility shift and co-purification assays. This confirms that direct and specific protein–protein interactions are responsible for the cooperativity observed between KorB and its corepressors and lays the basis for determining the biological importance of this cooperativity
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