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Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 151-157). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 74-82). Also available online in Scholars' Bank; and in ProQuest, free to University of Oregon users. Also available online.

Thesis (M.S.)--Villanova University, 2006. / Chemistry Dept. Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2005. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 75-82). Also available for download via the World Wide Web; free to University of Oregon users.

The advancement of catalytic systems and the application thereof has proven to be the key to overcome traditional limitations of industrial-scale synthetic processes. Converging organometallic and biocatalytic principles lead to the development of Artificial Metalloenzymes (ArMs) that comprise a synthetic metal catalyst embedded in a protein scaffold, thereby combining the reactivity of the former with the versatility of the latter. This synergistic approach introduces rationally designed building blocks for the catalytic site and the host protein to assemble enzyme-like structures that follow regio-, chemo-, enantio- and substrate-selective principles. Yet, the identification of suitable protein scaffolds has thus far been challenging. Herein we report a rationally optimized fluorescent protein host, mTFP*, that was engineered to have no intrinsic metal binding capability and, owing to its robust nature, can act as scaffold for the design of novel ArMs. We demonstrate the potential of site-specific modifications within the protein host, use protein X-Ray analysis to validate the respective scaffolds and show how artificial mutant binding sites can be introduced. Transition metal Förster Resonance Energy transfer (tmFRET) methodologies help to evaluate micromolar dissociation constants and reveal structural rearrangements upon coordination of the metal centers. In conjunction with molecular insights from X-Ray crystallographic structure determination, dynamics of the binding pocket can be inferred. The versatile subset of different binding motifs paired with transition metal catalysts create artificial metalloenzymes that provide reactivities which otherwise do not exist in nature. As a proof of concept, Diels-Alder cycloadditions highlight the potential of the present mTFP* based catalysts by stereoselectively converting azachalcone and cyclopentadiene substrates. Screens indicate an enantiomeric excess of up to 60% and provide insights into the electronic and geometric constitution of the first coordination spheres binding the catalysts. We further apply two general principles to optimize selective conversions of the generated ArMs. 1) Utilizing site-specific mutagenesis, increased hydrophobicity is introduced to the second coordination sphere. 2) In-vitro post-expressional modification utilizing N-hydroxysuccinimide esters is anticipated to introduce a sterically more demanding second coordination sphere that influences substrate entry by favoring a particular stereoisomer. The latter approach however also enhances the host proteins robustness under processing conditions. The presented study investigates a novel approach to create artificial metalloenzymes based on non-enzymatic precursor proteins. It illustrates means of modification and functionalization. Further guidance to overcome the general problem of insufficient stereoselectivity and stability is also presented. In view of the insights gained we see the importance of further mutagenic studies, i.e. through means of guided evolution, to extend stereoselectivities. In-vivo applications of artificial metalloenzymes could thus be used to pursue metabolomic engineering.

Green fluorescent protein (GFP) fusion tags are commonly used to study protein expression and cellular localization in vivo. But, GFP must undergo an autogenic post-translational modification, known as chromophore maturation, to become fluorescent, a process that can have a half-time longer than 30 minutes inside research model organisms. The timescale of chromophore maturation in GFP is thus slower than many key biological processes, limiting its usefulness in measuring those processes. In this thesis, we discuss the creation and engineering of a sensor for transiently expressed proteins (STEP) based on a fully matured but dim GFP. Upon specific binding of STEPtag, a small (15.5 kDa) protein to the sensor, full fluorescence is restored. Thus, by genetically fusing STEPtag to a protein of interest, it can be detected as soon as folding is complete, without any maturation delay. Through a combination of rational design and targeted directed evolution, we describe the improvement of the original sensor, gSTEP0, into an optimized version, gSTEP1. The sensor has been validated in vitro and in E. coli cells, and we have found that for gSTEP1, the fluorescence signal increases more than three-fold upon binding, with a Kd of 120 ± 30 nM and a kon of 1.7 x 105 M-1s-1, allowing detection of the protein of interest on the second timescale. We have also created a yellow version of the biosensor, and provide preliminary attempts at developing orthogonal binding pairs, as well as red- and cyan-coloured STEPs, which could eventually be used in multiplex experiments. Our biosensor opens the door to the study of short-timescale processes in research model organisms, such as Drosophila and zebrafish embryogenesis, as well as in host-pathogen interactions, which we are currently investigating.

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