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1.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 12): 1270-82, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19966413

ABSTRACT

A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 A resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested.


Subject(s)
Bacterial Proteins/chemistry , Deinococcus/chemistry , Ribosomal Proteins/chemistry , Ribosome Subunits, Large, Bacterial/chemistry , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Cell Fractionation , Crystallography, X-Ray , Deinococcus/genetics , Deinococcus/growth & development , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Structure, Quaternary , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , Ribosomal Proteins/isolation & purification , Ribosome Subunits, Large, Bacterial/genetics
2.
Anal Biochem ; 395(1): 77-85, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19646947

ABSTRACT

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.


Subject(s)
Deinococcus/ultrastructure , Ribosome Subunits, Large, Bacterial/chemistry , Bacterial Proteins/analysis , Centrifugation, Density Gradient , Chromatography, Affinity , Chromatography, High Pressure Liquid , Cloning, Molecular , Databases, Protein , Gene Expression , Magnesium Chloride , Oligopeptides , Peptide Fragments/analysis , Peptides/genetics , RNA, Bacterial/analysis , RNA, Ribosomal/analysis , Recombinant Fusion Proteins , Ribosomal Proteins/analysis , Ribosomal Proteins/genetics , Ribosome Subunits, Large, Bacterial/metabolism , Ribosome Subunits, Small, Bacterial/genetics , Ribosome Subunits, Small, Bacterial/metabolism , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry
3.
Antimicrob Agents Chemother ; 53(2): 496-504, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19047657

ABSTRACT

By using a detergent-washed membrane preparation, the interaction of the fungal natural product inhibitor aureobasidin A (AbA) with inositol phosphorylceramide synthase (IPC synthase) was studied by kinetic analysis of wild-type and mutant enzyme-catalyzed reactions. AbA inhibited the wild-type enzyme from both Candida albicans and Saccharomyces cerevisiae in an irreversible, time-dependent manner, with apparent K(i) values of 183 and 234 pM, respectively. Three synthetic chemistry-derived AbA derivatives, PHA-533179, PHA-556655, and PHA-556656, had affinities 4 to 5 orders of magnitude lower and were reversible inhibitors that competed with the donor substrate phosphatidylinositol (PI). AbA was a reversible, apparently noncompetitive inhibitor, with a K(i) of 1.4 microM, of the IPC synthase from an AbA-resistant S. cerevisiae mutant. The K(m) values for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the V(max) for the mutant enzyme was less than 10% of that for the wild-type enzyme. A comparison of the results obtained with AbA with those obtained with two other natural products inhibitors, rustmicin and khafrefungin, revealed that while rustmicin appeared to be a reversible, noncompetitive inhibitor of the wild-type enzyme, with a K(i) of 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent K(i) of 0.43 nM. An evaluation of the efficiencies of these compounds as inhibitors of the mutant enzyme revealed for both a drop in the apparent affinity for the enzyme of more than 2 orders of magnitude.


Subject(s)
Antifungal Agents/pharmacology , Depsipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Hexosyltransferases/antagonists & inhibitors , Algorithms , Candida albicans/drug effects , Drug Resistance, Fungal , Glycolipids/pharmacology , Kinetics , Saccharomyces cerevisiae/drug effects , Time Factors
4.
Proc Natl Acad Sci U S A ; 105(36): 13309-14, 2008 Sep 09.
Article in English | MEDLINE | ID: mdl-18757755

ABSTRACT

The phosphodiesterases (PDEs) are metal ion-dependent enzymes that regulate cellular signaling by metabolic inactivation of the ubiquitous second messengers cAMP and cGMP. In this role, the PDEs are involved in many biological and metabolic processes and are proven targets of successful drugs for the treatments of a wide range of diseases. However, because of the rapidity of the hydrolysis reaction, an experimental knowledge of the enzymatic mechanisms of the PDEs at the atomic level is still lacking. Here, we report the structures of reaction intermediates accumulated at the reaction steady state in PDE9/crystal and preserved by freeze-trapping. These structures reveal the catalytic process of a PDE and explain the substrate specificity of PDE9 in an actual reaction and the cation requirements of PDEs in general.


Subject(s)
Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/metabolism , Catalysis , Crystallography, X-Ray , Guanine Nucleotides/chemistry , Guanine Nucleotides/metabolism , Humans , Hydrolysis , Kinetics , Models, Molecular , Mutation/genetics , Phosphoric Diester Hydrolases/genetics , Protein Structure, Tertiary , Substrate Specificity
5.
Biochemistry ; 43(26): 8483-93, 2004 Jul 06.
Article in English | MEDLINE | ID: mdl-15222759

ABSTRACT

The effect of 26 different membrane-perturbing agents on the activity and phase distribution of inositol phosphorylceramide synthase (IPC synthase) activity in crude Candida albicans membranes was investigated. The nonionic detergents Triton X-100, Nonidet P-40, Brij, Tween, and octylglucoside all inactivated the enzyme. However, at moderate concentrations, the activity of the Triton X-100- and octylglucoside-solubilized material could be partially restored by inclusion of 5 mM phosphatidylinositol (PI) in the solubilization buffer. The apparent molecular mass of IPC synthase activity solubilized in 2% Triton X-100 was between 1.5 x 10(6) and 20 x 10(6) Da, while under identical conditions, octylglucoside-solubilized activity remained associated with large presumably membrane-like structures. Increased detergent concentrations produced more drastic losses of enzymatic activity. The zwitterionic detergents Empigen BB, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), Zwittergent 3-10, and amidosulfobetaine (ASB)-16 all appeared capable of solubilizing IPC synthase. However, these agents also inactivated the enzyme essentially irreversibly. Solubilization with lysophospholipids again resulted in drastic losses of enzymatic activity that were not restored by the inclusion of PI. Lysophosphatidylinositol also appeared to compete, to some extent, with the donor substrate phosphatidylinositol. The sterol-containing agent digitonin completely inactivated IPC synthase. By contrast, sterol-based detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and taurodeoxycholate (tDOC) had little or no effect on the enzyme activity. The IPC synthase activity in C. albicans membranes remained largely intact and sedimentable at CHAPS concentrations (4%) where >90% of the phospholipids and 60% of the total proteins were extracted from the membranes. At 2.5% CHAPS, a concentration where approximately 50% of the protein and 80% of the phospholipids are solubilized, there was no detectable loss of enzyme activity, and it was found that the detergent-treated membranes had significantly improved properties compared to crude, untreated membranes as the source of IPC synthase activity. In contrast to assays utilizing intact membranes or Triton X-100 extracts, assays using CHAPS- or tDOC-washed membranes were found to be reproducible, completely dependent on added acceptor substrate (C(6)-7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-ceramide), and >95% dependent on added donor substrate (PI). Product formation was linear with respect to both enzyme concentration and time, and transfer efficiency was improved more than 20-fold as compared to assays using crude membranes. Determination of kinetic parameters for the two IPC synthase substrates using CHAPS-washed membranes resulted in K(m) values of 3.3 and 138.0 microM for C(6)-NBD-ceramide and PI, respectively. In addition, the donor substrate, PI, was found to be inhibitory at high concentrations with an apparent K(i) of 588.2 microM.


Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Biochemistry/methods , Candida albicans/enzymology , Hexosyltransferases/chemistry , 4-Chloro-7-nitrobenzofurazan/pharmacology , Butyrates/pharmacology , Centrifugation, Density Gradient , Ceramides/pharmacology , Cetomacrogol/pharmacology , Cholic Acids/pharmacology , Chromatography , Detergents/pharmacology , Dose-Response Relationship, Drug , Glucosides/pharmacology , Hexosyltransferases/metabolism , Kinetics , Lipids/chemistry , Lysophospholipids/chemistry , Octoxynol/pharmacology , Phospholipids/metabolism , Polidocanol , Polyethylene Glycols/pharmacology , Polysorbates/pharmacology , Quaternary Ammonium Compounds/pharmacology , Taurodeoxycholic Acid/pharmacology , Time Factors
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