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Title:Probing the roles of metal binding ligands in cupredoxins: incorporating nonproteinogenic amino acids into azurin and CuA Azurin
Author(s):Clark, Kevin M.
Director of Research:Lu, Yi; van der Donk, Wilfred A.
Doctoral Committee Chair(s):Lu, Yi
Doctoral Committee Member(s):van der Donk, Wilfred A.; Jakobsson, Eric; Crofts, Antony R.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
CuA Azurin
Expressed Protein Ligation
backbone ester
peptide ligation
Abstract:Metalloproteins play important roles in biological systems since metal-binding sites are found in ~ 1/3 of structurally characterized proteins and in ~ 1/2 of all proteins. Despite progress, the role of these metal-binding sites remains poorly understood. Furthermore, their detailed geometric and electronic structure has yet to be fully elucidated. Site-directed mutagenesis using natural amino acids has continued to be the primary method of probing of metal-binding sites. However, it has become increasingly evident that site-directed mutagenesis, though powerful, cannot address the precise role of the metal ligands without changing multiple factors because of the relative paucity of functional group availability within the proteinogenic amino acids. To address the need to precisely determine specific metal binding ligand functionalities, and further efforts to engineer new function into proteins, Expressed Protein Ligation (EPL) has been used to introduce non-proteinogenic amino acids to metalloproteins. Using EPL to specifically probe the Type 1 (T1) copper site of azurin and the CuA site found in engineered CuA Azurin has aided the understanding of the roles of each ligand in this electron transfer protein. This thesis covers the replacement of two ligands in azurin, Cys112 and Met121, with nonproteinogenic amino acid analogues. Investigating the Cys112(S)-Cu Ligand-to-Metal Charge Transfer (LMCT) that gives azurin its intense blue color and its signature absorbance at 625 nm in the visible spectrum using two different analogues has been performed. Incorporating selenocysteine for Cys112 resulted in a new variant that maintained type 1 geometry and will with future experiments, help to elucidate the electron density of the site with more precision. To further expand this concept, EPL was used to incorporate homocysteine (Hcy) to investigate the rack hypothesis. Insertion of a Cys derivative with an additional methylene group demonstrated that the type 1 site in azurin was capable of adjusting sufficiently through protein folding to maintain type 1 geometry and function. Together, these mutations provide insight into the unique relationship between the site’s geometric features and its electronic features by minimally perturbing the site and selectively altering each feature individually. Secondly, while conversion of blue copper sites with a weak axial ligand to green copper sites containing a medium strength axial ligand has been demonstrated in cupredoxins, converting blue copper sites to a red copper site with a strong axial ligand has not been reported. By replacing weaker thiolate from Met121 in azurin with the stronger thiolate ligand from Hcy, a variant was created whose spectra (RL= 1.5, and A|| = 180 x 10-4 cm-1) and Cu-S(Cys) distance (2.22 Å) were very similar to those of the red copper protein nitrosocyanin. The results strongly support the “coupled distortion” model that helps explain axial ligand tuning of spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino acids to address critical chemical biological questions. Lastly, the unique Cu coordination of a backbone carbonyl in CuA azurin site was investigated using backbone carbonyl analogues of Glu. By replacing the carbonyl of the natural peptide linkage with an ester moiety, we have shown that the nucleophilicity of the carbonyl does not seem to play a major role in either the spectroscopic or reduction potential features of the CuA site. These studies have provided a unique methodology to investigate metal ligand interactions outside of the standard side chain interactions. In summary a means to investigate the specific function of individual metal ligand residues in azurin and CuA azurin by using EPL to incorporate nonproteinogenic amino acid analogues has been demonstrated. This work has resulted in new insights into the role of the metal ligands and has shaped future metalloprotein engineering efforts.
Issue Date:2010-08-31
Rights Information:Copyright 2010 Kevin M. Clark
Date Available in IDEALS:2010-08-31
Date Deposited:2010-08

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