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Title:Toward Engineering Oxygenase Activity into the Electron Transfer Protein Azurin
Author(s):Sieracki, Nathan A.
Director of Research:Lu, Yi
Doctoral Committee Chair(s):Lu, Yi
Doctoral Committee Member(s):Suslick, Kenneth S.; Gennis, Robert B.; Hartwig, John F.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
sulfenic acid
TIP buffer
Abstract:Structural knowledge of the metal environment is important in understanding the function of metalloproteins and is critical to harnessing their power. Modeling within a protein scaffold offers a pathway to the rational design of desired function s by use of Nature’s own tools; both to gain novel insights into metal-mediated processes and to create new processes. The broad goal of this work is successful engineering of monooxygenase function into model proteinic scaffolds. We seek to model within a prototype scaffold the function of the PHM (peptidylyglycine--hydroxylating monooxygenase) and DβM (dopamine -monooxygenase) family of enzymes, which are capable of remarkable C-H bond activation under ambient conditions with ascorbic acid and molecular oxygen. In Chapter 2, the power of the cavity mutant approach to small molecule processing in the azurin scaffold is demonstrated through description of the first reported Cu(II)-sulfenic acid species. This species, prepared in high yield through copper mediated reduction of hydrogen peroxide, represents the first reported chemical transformation in ‘converted’ electron transfer protein. Furthermore, it is the first report of a synthetic sulfenic acid functionality within the context of a protein. The latter is an important contribution to the field of protein design, as precise knowledge and control of cysteine oxidation state is an important parameter in dictating function in cysteine-containing native and designed peptides and proteins. In Chapter 3, a series of Type-2 azurin variants is reported in which the anionic redox ‘chameleon’ Cys-112 is replaced with neutral His-112 in an effort to develop a robust protein scaffold for modeling of substrate C-H bond activation. A series of surrogate, intramolecular substrates that offer an accessible H-atom were generated through site directed mutagenesis at the axial Met-121 position above the trigonal plane. The spectral characterization of the series is described, and mutant-dependent reactivity with added O2 and ascorbic acid is described. In Chapter 4, the fatty-acid carrier protein, Cellular Retinoic Acid Binding Protein (CRAPB-II) is described in the context of hosting small molecule catalysts. Initial investigations into the design of a binding site for the carbene transfer catalyst, rhodium acetate, will be described, as well as investigation into the utility of the CRABP-II scaffold for aqueous nitric oxide sensing. Finally, in Chapter 5 will be discussed the development of a temperature independent pH buffer, along with demonstration of its utility in low-temperature antibiotic storage, and low temperature spectroscopy.
Issue Date:2011-01-21
Rights Information:Copyright 2010 Nathan A. Sieracki
Date Available in IDEALS:2011-01-21
Date Deposited:2010-12

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