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1.  Recombinant production, crystallization and X-ray crystallographic structure determination of the peptidyl-tRNA hydrolase of Pseudomonas aeruginosa  
P. aeruginosa peptidyl-tRNA hydrolase was cloned, expressed, purified and crystallized. The crystals obtained diffracted to 1.77 Å resolution.
The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6122 with unit-cell parameters a = b = 63.62, c = 155.20 Å, α = β = 90, γ = 120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å3 Da−1. The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.
doi:10.1107/S1744309112045770
PMCID: PMC3509967  PMID: 23192026
peptidyl-tRNA hydrolases; peptidyl-tRNA; Pseudomonas aeruginosa
2.  Structure of the endonuclease IV homologue from Thermotoga maritima in the presence of active-site divalent metal ions 
When compared with their E. coli homologue, two X-ray crystal structures of T. maritima endonuclease IV differing in the composition of the trinuclear metal site point to the importance of the trinuclear site and its modulation among species to the function of this enzyme in the AP endonuclease IV family.
The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-­hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically, Thermotoga maritima endonuclease IV is a member of the second conserved AP endonuclease family that includes Escherichia coli endonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-­ray crystal structures of the T. maritima endonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of the T. maritima endonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.
doi:10.1107/S1744309110028575
PMCID: PMC2935215  PMID: 20823514
apurinic/apyrimidinic endonucleases; endonuclease IV; DNA-repair proteins; Thermotoga maritima
3.  Purification, crystallization and preliminary crystallographic analysis of a thermostable endonuclease IV from Thermotoga maritima  
The overexpression, purification and crystallization of endonuclease IV from T. maritima are reported. The crystals belonged to the hexagonal space group P61 and diffracted to 2.36 Å resolution.
The DNA-repair enzyme endonuclease IV from the thermophilic bacterium Thermotoga maritima MSB8 (reference sequence NC_000853) has been expressed in Escherichia coli and crystallized for X-ray analysis. T. maritima endonuclease IV is a 287-amino-acid protein with 32% sequence identity to E. coli endonuclease IV. The protein was purified to homogeneity and was crystallized using the sitting-drop vapor-diffusion method. The protein crystallized in space group P61, with one biological molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.39 Å3 Da−1 and 47% solvent content. The unit-cell parameters of the crystals were a = b = 123.2, c = 35.6 Å. Microseeding and further optimization yielded crystals with an X-ray diffraction limit of 2.36 Å. A single 70° data set was collected and processed, resulting in an overall R merge and a completeness of 9.5% and 99.3%, respectively.
doi:10.1107/S1744309109047393
PMCID: PMC2802891  PMID: 20054139
endonuclease IV; DNA-repair proteins; Thermotoga maritima
4.  Preliminary neutron and ultrahigh-resolution X-ray diffraction studies of the aspartic proteinase endothiapepsin cocrystallized with a gem-diol inhibitor 
Three data sets have been collected on endothiapepsin complexed with the gem-diol inhibitor PD-135,040: a high-resolution synchrotron X-ray data set, a room-temperature X-ray data set and a neutron diffraction data set. Until recently, it has been impossible to grow large protein crystals of endothiapepsin with any gem-diol inhibitor that are suitable for neutron diffraction.
Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme–inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.
doi:10.1107/S1744309107061283
PMCID: PMC2344097  PMID: 18084100
endothiapepsin; gem-diol inhibitors; neutron diffraction
5.  Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of d-xylose isomerase with bound d-xylulose†∞ 
Biochemistry  2008;47(29):7595-7597.
The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme D-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, D-xylulose, shows, unexpectedly, that O5 of D-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in close proximity to C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.
doi:10.1021/bi8005434
PMCID: PMC2488401  PMID: 18578508
7.  Preliminary neutron and ultrahigh-resolution X-ray diffraction studies of the aspartic proteinase endothiapepsin cocrystallized with a gem-diol inhibitor 
Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme—inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.
doi:10.1107/S1744309107061283
PMCID: PMC2344097  PMID: 18084100
8.  Synthesis, capillary crystallization and preliminary joint X-ray and neutron crystallographic study of Z-­DNA without polyamine at low pH 
Joint X-ray and neutron crystallographic data have been collected from the oligonucleotide d(CGCGCG) crystallized without polyamine and at low pH in order to study hydration in the protein-binding major groove of Z-DNA.
In order to crystallographically study the hydration of the major groove (convex surface) of Z-DNA, the oligonucleotide d(CGCGCG) has been synthesized. Single crystals were grown by vapor diffusion using the hanging-drop and sitting-drop methods for X-ray studies and by batch crystallization and evaporation within silicon tubes for neutron studies. Hexagonal crystals were obtained without the use of duplex-stabilizing polyamines and at an acid pH. X-­ray data collected at room temperature (1.5 Å resolution; unit-cell parameters a = 17.90, b = 30.59, c = 44.61 Å) and at 100 K (1 Å resolution; a = 17.99, b = 30.98, c = 44.07 Å) and neutron data collected at room temperature (1.6 Å resolution; a = 18.00, b = 31.16, c = 44.88 Å) indicate that the DNA is in the Z-form packing in space group P212121.
doi:10.1107/S174430910601236X
PMCID: PMC2219980  PMID: 16682774
d(CGCGCG); Z-DNA; hydration
9.  Protein structures by spallation neutron crystallography 
Journal of Synchrotron Radiation  2008;15(Pt 3):215-218.
The capabilities of the Protein Crystallography Station at Los Alamos Neutron Science Center for determining protein structures by spallation neutron crystallography are illustrated, and the methodological and technological advances that are emerging from the Macromolecular Neutron Crystallography consortium are described.
The Protein Crystallography Station at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. This capability also includes the Macromolecular Neutron Crystallography (MNC) consortium between Los Alamos (LANL) and Lawrence Berkeley National Laboratories for developing computational tools for neutron protein crystallography, a biological deuteration laboratory, the National Stable Isotope Production Facility, and an MNC drug design consortium between LANL and Case Western Reserve University.
doi:10.1107/S0909049508000824
PMCID: PMC2394804  PMID: 18421142
neutrons; proteins; macromolecular crystallography; deuteration; enzyme mechanisms; drug binding; hydration; joint XN structure refinement
10.  A Structural Study of Norovirus 3C Protease Specificity: Binding of a Designed Active Site-Directed Peptide Inhibitor† 
Biochemistry  2010;50(2):240-249.
Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 Å resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics.
doi:10.1021/bi1008497
PMCID: PMC3058531  PMID: 21128685

Results 1-10 (10)