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Full-Text Articles in Physics
Potentially Diagnostic Electron Paramagnetic Resonance Spectra Elucidate The Underlying Mechanism Of Mitochondrial Dysfunction In The Deoxyguanosine Kinase Deficient Rat Model Of A Genetic Mitochondrial Dna Depletion Syndrome, Brian Bennett, Daniel Helbling, Hui Meng, Jason Jarzembowski, Aron M. Geurts, Marisa W. Friederich, Johan L.K. Van Hove, Michael W. Lawlor, David P. Dimmock
Potentially Diagnostic Electron Paramagnetic Resonance Spectra Elucidate The Underlying Mechanism Of Mitochondrial Dysfunction In The Deoxyguanosine Kinase Deficient Rat Model Of A Genetic Mitochondrial Dna Depletion Syndrome, Brian Bennett, Daniel Helbling, Hui Meng, Jason Jarzembowski, Aron M. Geurts, Marisa W. Friederich, Johan L.K. Van Hove, Michael W. Lawlor, David P. Dimmock
Physics Faculty Research and Publications
A novel rat model for a well-characterized human mitochondrial disease, mitochondrial DNA depletion syndrome with associated deoxyguanosine kinase (DGUOK) deficiency, is described. The rat model recapitulates the pathologic and biochemical signatures of the human disease. The application of electron paramagnetic (spin) resonance (EPR) spectroscopy to the identification and characterization of respiratory chain abnormalities in the mitochondria from freshly frozen tissue of the mitochondrial disease model rat is introduced. EPR is shown to be a sensitive technique for detecting mitochondrial functional abnormalities in situ and, here, is particularly useful in characterizing the redox state changes and oxidative stress that can result …
Moving Difference (Mdiff) Non-Adiabatic Rapid Sweep (Nars) Epr Of Copper(Ii), James S. Hyde, Brian Bennett, Aaron W. Kittell, Jason M. Kowalski, Jason Walter Sidabras
Moving Difference (Mdiff) Non-Adiabatic Rapid Sweep (Nars) Epr Of Copper(Ii), James S. Hyde, Brian Bennett, Aaron W. Kittell, Jason M. Kowalski, Jason Walter Sidabras
Physics Faculty Research and Publications
Non-adiabatic rapid sweep (NARS) EPR spectroscopy has been introduced for application to nitroxide-labeled biological samples (Kittell et al., 2011). Displays are pure absorption, and are built up by acquiring data in spectral segments that are concatenated. In this paper we extend the method to frozen solutions of copper-imidazole, a square planar copper complex with four in-plane nitrogen ligands. Pure absorption spectra are created from concatenation of 170 5-gauss segments spanning 850 G at 1.9 GHz. These spectra, however, are not directly useful since nitrogen superhyperfine couplings are barely visible. Application of the moving difference (MDIFF) algorithm to the digitized NARS …
Arabidopsis Thaliana Glx2-1 Contains A Dinuclear Metal Binding Site, But Is Not A Glyoxalase 2, Pattraranee Limphong, Michael W. Crowder, Brian Bennett, Christopher A. Makaroff
Arabidopsis Thaliana Glx2-1 Contains A Dinuclear Metal Binding Site, But Is Not A Glyoxalase 2, Pattraranee Limphong, Michael W. Crowder, Brian Bennett, Christopher A. Makaroff
Physics Faculty Research and Publications
In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV–visible, EPR, and 1H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV–visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over …
Analyzing The Catalytic Role Of Asp97 In The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, Richard C. Holz
Analyzing The Catalytic Role Of Asp97 In The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma–atomic emission spectroscopy analysis revealed that 1.0 ± 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ∼ 9000-fold decrease in kca towards …
X-Ray Crystallographic Characterization Of The Co(Ii)-Substituted Tris-Bound Form Of The Aminopeptidase From Aeromonas Proteolytica, Petra Munih, Aaron Moulin, Carin Stamper, Brian Bennett, Dagmar Ringe, Gregory A. Petsko, Richard C. Holz
X-Ray Crystallographic Characterization Of The Co(Ii)-Substituted Tris-Bound Form Of The Aminopeptidase From Aeromonas Proteolytica, Petra Munih, Aaron Moulin, Carin Stamper, Brian Bennett, Dagmar Ringe, Gregory A. Petsko, Richard C. Holz
Physics Faculty Research and Publications
The X-ray crystal structure of the Co(II)-loaded form of the aminopeptidase from Aeromonas proteolytica ([CoCo(AAP)]) was solved to 2.2 Å resolution. [CoCo(AAP)] folds into an α/β globular domain with a twisted β-sheet hydrophobic core sandwiched between α-helices, identical to [ZnZn(AAP)]. Co(II) binding to AAP does not introduce any major conformational changes to the overall protein structure and the amino acid residues ligated to the dicobalt(II) cluster in [CoCo(AAP)] are the same as those in the native Zn(II)-loaded structure with only minor perturbations in bond lengths. The Co(II)–Co(II) distance is 3.3 Å. Tris(hydroxymethyl)aminomethane (Tris) coordinates to the dinuclear Co(II) active site …
Experimental Evidence For A Metallohydrolase Mechanism In Which The Nucleophile Is Not Delivered By A Metal Ion: Epr Spectrokinetic And Structural Studies Of Aminopeptidase From Vibrio Proteolyticus, Amit Kumar, Gopal R. Periyannan, Aaron W. Kittell, Jung Ja Kim, Brian Bennett
Experimental Evidence For A Metallohydrolase Mechanism In Which The Nucleophile Is Not Delivered By A Metal Ion: Epr Spectrokinetic And Structural Studies Of Aminopeptidase From Vibrio Proteolyticus, Amit Kumar, Gopal R. Periyannan, Aaron W. Kittell, Jung Ja Kim, Brian Bennett
Physics Faculty Research and Publications
Metallohydrolases catalyse some of the most important reactions in biology and are targets for numerous chemotherapeutic agents designed to combat bacterial infectivity, antibiotic resistance, HIV infectivity, tumour growth, angiogenesis and immune disorders. Rational design of inhibitors of these enzymes with chemotherapeutic potential relies on detailed knowledge of the catalytic mechanism. The roles of the catalytic transition ions in these enzymes have long been assumed to include the activation and delivery of a nucleophilic hydroxy moiety. In the present study, catalytic intermediates in the hydrolysis of L-leucyl-L-leucyl-L-leucine by Vibrio proteolyticus aminopeptidase were characterized in spectrokinetic and structural studies. Rapid-freeze-quench EPR studies …