Browsing by Subject "Azurin"

Now showing 1 - 5 of 5
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    The incorporation of nitrosocyanin copper binding loop into azurin.
    (2010-07) Schenewerk, Audrey Rose
    Metalloprotein design and engineering can be used to probe our understanding of active site structure and function. Loop-directed mutagenesis has been used in the metalloprotein field to change the copper binding loops from a number of members of the cupredoxin family into other protein scaffolds. We report the replacement of a ten amino acid loop that supports the copper binding site in the blue copper protein azurin with the red copper binding loop from the protein nitrosocyanin. Azurin is an electron transfer protein while the role of nitrosocyanin is unknown, yet believed to be catalytic. In addition to the loop, we added a carboxylic acid residue into the copper binding site which fully models the site of nitrosocyanin. Synthesis, expression, and UV-visible absorption and EPR spectra for this series of azurin variants will be reported.
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    Investigation and Identification of Novel Hyperthermophilic Manganese(II)-Oxidase from Purple Pool Yellowstone National Park
    (2022-10) Tozaki, Naoto
    Manganese (Mn) oxides are some of the strongest naturally occurring oxides in nature, playing an important role in geochemical cycling, and living systems. Several theories on the role of Mn in microbial systems exist, but there is not enough supporting evidence to provide a strong argument for these theories. Understanding the role of Mn in microbial systems will allow for further investigations into the evolution of early life, defensive mechanisms, and electron transfer systems in microbes. Preliminary sequence data of biofilms and enrichments of biofilms collected from a Mn-depositing hot spring (102ºC) in Yellow Stone National Park (YNP), have yielded findings that suggest a novel hyperthermophilic archaea may be responsible for the accumulation of Mn(III/IV) oxides within the hot spring. There are currently no known hyperthermophilic archaea that is capable of oxidizing Mn(II), an investigation into the properties of the Mn oxidizing properties will have wide impacts in fields of early life, bioremediation, and industry. The investigation will involve biofilm studies, to study its role in Mn oxidation mechanisms, and also in Mn oxide morphologies as a respect of Mn concentration and flow rate. These studies would be useful in biomarker analysis in Mn containing geological features, most notably in YNP. A bioinformatics component of the investigation will involve an investigation of the genome and mapping of possible gene sequences responsible for Mn oxidation. The Mn oxidase genes will then be further contextualized by constructing metabolic pathways of the genomes. The biochemical component will include the isolation and expression, and synthesis of the proteins from the sequenced identified for Mn oxidation. As a protein originating from hyperthermophilic archaea, it is hypothesized that it will have retained activity in high temperatures. These hypothesis will be tested by running activity assays, in respect to product/intermediate formations. In addition, denaturation conditions will be tested to examine the durability of the protein structure. Characterization of the novel Mn oxidation components of the thermophilic archaea will be impactful in fields of environmental science, bioinorganic chemistry, and industry.
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    Nitrite Reductase Activities of Azurin Variants
    (2015-08) Khatiwada, Balabhadra
    Nitrite reductase (NiR) catalyzes the reduction of nitrite to nitric oxide in the denitrification stage of nitrogen cycle. To study the structure and function of this dinuclear copper enzyme, mimics were designed by adding a second copper-binding site on the surface of mononuclear copper protein azurin. The existing type1 (T1) copper-binding site in azurin is structurally and functionally similar to that of NiR. T1 site was maintained in azurin whereas an additional catalytic type2 (T2) site, similar to the T2 site of NiR, was designed. The designed T2 site in azurin mimics the spectroscopic and functional aspects of NiR. The UV-Vis absorption and EPR spectra of azurin variants showed similarities to that of native NiR. We further examined the activity of our variants using the activity assays. Reduction and re-oxidation assays of T1 site in azurin variants were determined and compared with variants with modified T1 copper center redox potentials.
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    Purification, crystallization and structure determination of an azurin variant.
    (2012-08) Ladd, Melanie Ann
    X-ray diffraction is a method that allows the three-dimensional structure of a molecule to be determined. To use this technique to study a protein model, high-quality crystals were grown. A biosynthetic approach was taken to model the mammalian protein peptidylglycine α-hydroxylating monooxygenase (PHM), which is a copper-binding protein that hydroxylates the α-carbon of a glycine residue in the production of peptide hormones. In order to understand the mechanism of this reaction, a model of the two copper sites involved in hydroxylation was created using the bacterial protein azurin as a scaffold (Az-PHM). To compare the structural similarity of the model to the native PHM system, Az-PHM crystals were grown for x-ray diffraction using various buffers, salts, polyethylene glycol (PEG) and excess copper. Dozens of the resulting crystals were diffracted, which had lower resolutions (~2.5 Å) and higher mosaicities (0.8 - 1.2° on average). Crystal dehydration and cryoprotection techniques were applied and consistently yielded higher resolution and lower mosaicity crystals. The crystal with the highest resolution and low mosaicity was grown in Tris buffer, lithium nitrate, PEG-2000 and copper chloride. Diffraction images for this crystal were collected on a Rigaku RAPID II X-ray Diffractometer using a copper radiation source with capillary optics and an R-AXIS image plate detector. Data were indexed to yield a P212121 space group, which was then followed by integration, scaling and averaging using CrystalClear 2.1 software. Phases were determined using the Molecular Replacement method in the software CCP4. Finally, structural refinement of the model and electron density map in Coot yielded a 1.3 Å structure with an Rfactor of 17.57% and an Rfree of 20.70%.
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    Rational improvement of the activity of a nitrite reductase model
    (2014-05) Strange, Jacob
    Nitrite reductase (NiR) is a bacterial enzyme that catalyzes the one electron reduction of nitrite (NO2-) to nitric oxide (NO). Through site-directed PCR mutagenesis, variants of the electron transfer protein azurin (See figure) were rationally designed to mimic Nitrite Reductase (NiR) in an attempt to increase our knowledge about enzyme function and design. Several variants were created by incorporation of a Type 2 copper center on the surface of the protein. Further mutations were added to aid in electron transfer as well as to match the potential of the Type 1 copper in azurin to that of the Type 1 copper in NiR. Each variant was characterized using EPR, UV-Vis and mass spectroscopy. Finally, we characterized each variant using Michaelis-Menten kinetics. This activity was determined using the Griess assay, which allows us to spectrophotometrically quantify the amount of nitrite in our reaction. We compared the activity of our NiR models. The results show that the variant designed for faster electron transfer did not increase the activity of our enzyme, but did increase activity when added with the other mutations. The two variants designed to decrease the reduction potential of the Type 1 copper site were found to increase the activity by themselves, but decrease the activity when combined.