Browsing by Author "Blackburn, G. Michael"

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    Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches
    (Wiley, 2017-08-07) Jin, Yi; Molt, Robert W.; Pellegrini, Erika; Cliff, Matthew J.; Bowler, Matthew W.; Richards, Nigel G. J.; Blackburn, G. Michael; Waltho, Jonathan P.; Biochemistry and Molecular Biology, School of Medicine
    We report X‐ray crystallographic and 19F NMR studies of the G‐protein RhoA complexed with MgF3 −, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The 19F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O3G and thence to PG. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that 19F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.
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    How to name atoms in phosphates, polyphosphates, their derivatives and mimics, and transition state analogues for enzyme-catalysed phosphoryl transfer reactions (IUPAC Recommendations 2016)
    (De Gruyter, 2017-05) Blackburn, G. Michael; Cherfils, Jacqueline; Moss, Gerard P.; Richards, Nigel G. J.; Waltho, Jonathan P.; Williams, Nicholas H.; Wittinghofer, Alfred; Chemistry and Chemical Biology, School of Science
    Procedures are proposed for the naming of individual atoms, P, O, F, N, and S in phosphate esters, amidates, thiophosphates, polyphosphates, their mimics, and analogues of transition states for enzyme-catalyzed phosphoryl transfer reactions. Their purpose is to enable scientists in very different fields, e.g. biochemistry, biophysics, chemistry, computational chemistry, crystallography, and molecular biology, to share standard protocols for the labelling of individual atoms in complex molecules. This will facilitate clear and unambiguous descriptions of structural results, as well as scientific intercommunication concerning them. At the present time, perusal of the Protein Data Bank (PDB) and other sources shows that there is a limited degree of commonality in nomenclature, but a large measure of irregularity in more complex structures. The recommendations described here adhere to established practice as closely as possible, in particular to IUPAC and IUBMB recommendations and to “best practice” in the PDB, especially to its atom labelling of amino acids, and particularly to Cahn-Ingold-Prelog rules for stereochemical nomenclature. They are designed to work in complex enzyme sites for binding phosphates but also to have utility for non-enzymatic systems. Above all, the recommendations are designed to be easy to comprehend and user-friendly.
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    Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes
    (Springer, 2017-04) Jin, Yi; Molt, Robert W., Jr.; Blackburn, G. Michael; Chemistry and Chemical Biology, School of Science
    The phosphoryl group, PO3-, is the dynamic structural unit in the biological chemistry of phosphorus. Its transfer from a donor to an acceptor atom, with oxygen much more prevalent than nitrogen, carbon, or sulfur, is at the core of a great majority of enzyme-catalyzed reactions involving phosphate esters, anhydrides, amidates, and phosphorothioates. The serendipitous discovery that the phosphoryl group could be labeled by "nuclear mutation," by substitution of PO3- by MgF3- or AlF4-, has underpinned the application of metal fluoride (MF x ) complexes to mimic transition states for enzymatic phosphoryl transfer reactions, with sufficient stability for experimental analysis. Protein crystallography in the solid state and 19F NMR in solution have enabled direct observation of ternary and quaternary protein complexes embracing MF x transition state models with precision. These studies have underpinned a radically new mechanistic approach to enzyme catalysis for a huge range of phosphoryl transfer processes, as varied as kinases, phosphatases, phosphomutases, and phosphohydrolases. The results, without exception, have endorsed trigonal bipyramidal geometry (tbp) for concerted, "in-line" stereochemistry of phosphoryl transfer. QM computations have established the validity of tbp MF x complexes as reliable models for true transition states, delivering similar bond lengths, coordination to essential metal ions, and virtually identical hydrogen bond networks. The emergence of protein control of reactant orbital overlap between bond-forming species within enzyme transition states is a new challenging theme for wider exploration.
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    Octahedral Trifluoromagnesate, an Anomalous Metal Fluoride Species, Stabilizes the Transition State in a Biological Motor
    (American Chemical Society, 2021-03-05) Ge, Mengyu; Molt, Robert W., Jr.; Jenkins, Huw T.; Blackburn, G. Michael; Jin, Yi; Antson, Alfred A.; Biochemistry and Molecular Biology, School of Medicine
    Isoelectronic metal fluoride transition state analogue (TSA) complexes, MgF3 - and AlF4 -, have proven to be immensely useful in understanding mechanisms of biological motors utilizing phosphoryl transfer. Here we report a previously unobserved octahedral TSA complex, MgF3(H2O)-, in a 1.5 Å resolution Zika virus NS3 helicase crystal structure. 19F NMR provided independent validation and also the direct observation of conformational tightening resulting from ssRNA binding in solution. The TSA stabilizes the two conformations of motif V of the helicase that link ATP hydrolysis with mechanical work. DFT analysis further validated the MgF3(H2O)- species, indicating the significance of this TSA for studies of biological motors.