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Articles 1 - 12 of 12
Full-Text Articles in Entire DC Network
Heterologous Expression And Purification Of Vibrio Proteolyticus (Aeromonas Proteolytica) Aminopeptidase: A Rapid Protocol, Mariam Hartley, Brian Bennett
Heterologous Expression And Purification Of Vibrio Proteolyticus (Aeromonas Proteolytica) Aminopeptidase: A Rapid Protocol, Mariam Hartley, Brian Bennett
Physics Faculty Research and Publications
Metalloaminopeptidases (mAPs) are enzymes that are involved in HIV infectivity, tumor growth and metastasis, angiogenesis, and bacterial infection. Investigation of structure–function relationships in mAPs is a prerequisite to rational design of anti-mAP chemotherapeutics. The most intensively studied member of the biomedically important dinuclear mAPs is the prototypical secreted Vibrio proteolyticus di-zinc aminopeptidase (VpAP). The wild-type enzyme is readily purified from the supernatant of cultures of V. proteolyticus, but recombinant variants require expression in Escherichia coli. A greatly improved system for the purification of recombinant VpAP is described. A VpAP-(His)6 polypeptide, containing an N-terminal propeptide, and a C-terminal …
Human Glyoxalase Ii Contains An Fe(Ii)Zn(Ii) Center But Is Active As A Mononuclear Zn(Ii) Enzyme, Pattraranee Limphong, Ross M. Mckinney, Nicole E. Adams, Brian Bennett, Christopher A. Makaroff, Thusitha Gunasekera, Michael W. Crowder
Human Glyoxalase Ii Contains An Fe(Ii)Zn(Ii) Center But Is Active As A Mononuclear Zn(Ii) Enzyme, Pattraranee Limphong, Ross M. Mckinney, Nicole E. Adams, Brian Bennett, Christopher A. Makaroff, Thusitha Gunasekera, Michael W. Crowder
Physics Faculty Research and Publications
Human glyoxalase II (Glx2) was overexpressed in rich medium and in minimal medium containing zinc, iron, or cobalt, and the resulting Glx2 analogues were characterized using metal analyses, steady-state and pre-steady-state kinetics, and NMR and EPR spectroscopies to determine the nature of the metal center in the enzyme. Recombinant human Glx2 tightly binds nearly 1 equiv each of Zn(II) and Fe. In contrast to previous reports, this study demonstrates that an analogue containing 2 equiv of Zn(II) cannot be prepared. EPR studies suggest that most of the iron in recombinant Glx2 is Fe(II). NMR studies show that Fe(II) binds to …
Analyzing The Binding Of Co(Ii)-Specific Inhibitors To The Methionyl Aminopeptidases From Escherichia Coli And Pyrococcus Furiosus, Sanghamitra Mitra, George Sheppard, Jieyi Wang, Brian Bennett, Richard C. Holz
Analyzing The Binding Of Co(Ii)-Specific Inhibitors To The Methionyl Aminopeptidases From Escherichia Coli And Pyrococcus Furiosus, Sanghamitra Mitra, George Sheppard, Jieyi Wang, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
Methionine aminopeptidases (MetAPs) represent a unique class of protease that is capable of the hydrolytic removal of an N-terminal methionine residue from nascent polypeptide chains. MetAPs are physiologically important enzymes; hence, there is considerable interest in developing inhibitors that can be used as antiangiogenic and antimicrobial agents. A detailed kinetic and spectroscopic study has been performed to probe the binding of a triazole-based inhibitor and a bestatin-based inhibitor to both Mn(II)- and Co(II)-loaded type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs. Both inhibitors were found to be moderate competitive inhibitors. The triazole-type inhibitor was found to …
Structure And Mechanism Of Copper- And Nickel-Substituted Analogues Of Metallo-Β-Lactamase L1, Zhenxin Hu, Lauren J. Spadafora, Christine E. Hajdin, Brian Bennett, Michael W. Crowder
Structure And Mechanism Of Copper- And Nickel-Substituted Analogues Of Metallo-Β-Lactamase L1, Zhenxin Hu, Lauren J. Spadafora, Christine E. Hajdin, Brian Bennett, Michael W. Crowder
Physics Faculty Research and Publications
In an effort to further probe metal binding to metallo-β-lactamase L1 (mβl L1), Cu- (Cu-L1) and Ni-substituted (Ni-L1) L1 were prepared and characterized by kinetic and spectroscopic studies. Cu-L1 bound 1.7 equiv of Cu and small amounts of Zn(II) and Fe. The EPR spectrum of Cu-L1 exhibited two overlapping, axial signals, indicative of type 2 sites with distinct affinities for Cu(II). Both signals indicated multiple nitrogen ligands. Despite the expected proximity of the Cu(II) ions, however, only indirect evidence was found for spin−spin coupling. Cu-L1 exhibited higher kcat (96 s−1) and Km (224 μM) values, as …
Epr Of Cu2+ Prion Protein Constructs At 2 Ghz Using The G⊥ Region To Characterize Nitrogen Ligation, James S. Hyde, Brian Bennett, Eric D. Walter, Glenn L. Millhauser, Jason Walter Sidabras, William E. Antholine
Epr Of Cu2+ Prion Protein Constructs At 2 Ghz Using The G⊥ Region To Characterize Nitrogen Ligation, James S. Hyde, Brian Bennett, Eric D. Walter, Glenn L. Millhauser, Jason Walter Sidabras, William E. Antholine
Physics Faculty Research and Publications
A double octarepeat prion protein construct, which has two histidines, mixed with copper sulfate in a 3:2 molar ratio provides at most three imidazole ligands to each copper ion to form a square-planar Cu2+ complex. This work is concerned with identification of the fourth ligand. A new (to our knowledge) electron paramagnetic resonance method based on analysis of the intense features of the electron paramagnetic resonance spectrum in the g⊥ region at 2 GHz is introduced to distinguish between three and four nitrogen ligands. The methodology was established by studies of a model system consisting of histidine imidazole …
Substrate Binding Preferences And PKA Determinations Of A Nitrile Hydratase Model Complex: Variable Solvent Coordination To [(Bmmp-Tasn)Fe]Otf, Martin G. O'Toole, Brian Bennett, Mark S. Mashuta, Craig A. Grapperhaus
Substrate Binding Preferences And PKA Determinations Of A Nitrile Hydratase Model Complex: Variable Solvent Coordination To [(Bmmp-Tasn)Fe]Otf, Martin G. O'Toole, Brian Bennett, Mark S. Mashuta, Craig A. Grapperhaus
Physics Faculty Research and Publications
The five-coordinate iron-dithiolate complex (N,N′-4,7-bis-(2′-methyl-2′-mercatopropyl)-1-thia-4,7-diazacyclononane)iron(III), [LFe]+, has been isolated as the triflate salt from reaction of the previously reported LFeCl with thallium triflate. Spectroscopic characterization confirms an S = 1/2 ground state in non-coordinating solvents with room temperature μeff = 1.78 μΒ and electron paramagnetic resonance (EPR) derived g-values of g1 = 2.04, g2 = 2.02 and g3 = 2.01. [LFe]+ binds a variety of coordinating solvents resulting in six-coordinate complexes [LFe-solvent]+. In acetonitrile the low-spin [LFe-NCMe]+ (g1 = 2.27, g2 = …
Mutation Of H63 And Its Catalytic Affect On The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Brian Bennett, Richard C. Holz
Mutation Of H63 And Its Catalytic Affect On The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
In order to gain insight into the mechanistic role of a flexible exterior loop near the active site, made up of Y62, H63, G64, and Y65, that has been proposed to play an important role in substrate binding and recognition in the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H63A enzyme was prepared. Mutation of H63 to alanine does not affect the ability of the enzyme to bind divalent metal ions. The specific activity of H63A EcMetAP-I was determined using four different substrates of varying lengths, namely, l-Met-p-NA, MAS, MGMM and MSSHRWDW. For the smallest/shortest …
The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae Contains Two Active-Site Histidine Residues, Danuta Gillner, David L. Bienvenue, Boguslaw P. Nocek, Andzrej Joachimiak, Vincentos Zachary, Brian Bennett, Richard C. Holz
The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae Contains Two Active-Site Histidine Residues, Danuta Gillner, David L. Bienvenue, Boguslaw P. Nocek, Andzrej Joachimiak, Vincentos Zachary, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT …
Antivortex Dynamics In Magnetic Nanostripes, Andrew Kunz, Eric C. Breitbach, Andy J. Smith
Antivortex Dynamics In Magnetic Nanostripes, Andrew Kunz, Eric C. Breitbach, Andy J. Smith
Physics Faculty Research and Publications
In a thin magnetic nanostripe, an antivortex nucleates inside a moving domain wall when driven by an in-plane magnetic field greater than the so-called Walker field. The nucleated antivortex must cross the width of the nanostripe before the domain wall can propagate again, leading to low average domain wall speeds. A large out-of-plane magnetic field, applied perpendicularly to the plane of the nanostripe, inhibits the nucleation of the antivortex leading to fast domain wall speeds for all in-plane driving fields. We present micromagnetic simulation results relating the antivortex dynamics to the strength of the out-of-plane field. An asymmetry in the …
Dependence Of Domain Wall Structure For Low Field Injection Into Magnetic Nanowires, Andrew Kunz, Sarah C. Reiff
Dependence Of Domain Wall Structure For Low Field Injection Into Magnetic Nanowires, Andrew Kunz, Sarah C. Reiff
Physics Faculty Research and Publications
Micromagnetic simulation is used to model the injection of a domain wall into a magnetic nanowire with field strengths less than the so-called Walker field. This ensures fast, reliable motion of the wall. When the wire is located at the edge of a small injecting disk, a bias field used to control the orientation of the domain wall can reduce the pinning potential of the structure. The low field injection is explained by a simple model, which relies on the topological nature of a domain wall. The technique can quickly inject multiple domain walls with a known magnetic structure.
Field Induced Domain Wall Collisions In Thin Magnetic Nanowires, Andrew Kunz
Field Induced Domain Wall Collisions In Thin Magnetic Nanowires, Andrew Kunz
Physics Faculty Research and Publications
In a two-dimensional magnetic nanowire, it is possible to engineer collisions between two domain walls put into motion by an externally applied field. We show that the topological defects that define the domain wall can be controlled to allow for both domain wall annihilation and preservation during the collisions as long as the wire remains thin. The preservation process can be used to release pinned domain walls from notches with small applied fields.
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 …