Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [147]-151).

Thesis (Ph.D.). / Written for the Dept. of Biomedical Engineering. Title from title page of PDF (viewed 2009/06/18). Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (January 11, 2006) 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.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Fahrni, Christoph, Committee Chair ; Doyle, Donald, Committee Member ; Wilkinson, Angus, Committee Member ; Zhang, Z. John, Committee Member ; Radhakrishna, Harish, Committee Member.

This research project focuses on the synthesis of fluorescent polymers with pendant triazole-substituted coumarin side chain units. Copper(I)-catalyzed Huisgen’s 1,3-dipolar cycloaddition of alkynes with azides to form a 1,2,3-triazole ring, a typical example of “click” reaction, has been utilized for the synthesis of a novel vinyl monomer, 2-oxo-3-(4- vinyl-1H-1,2,3-triazol-1-yl)-2H-chromen-7-yl acetate 62. The monomer and its precursors were synthesised and characterized using 1D- and 2D-NMR and FT-IR. Coumarin-containing triazole polymers were synthesised using free radical polymerization. Reversible Addition-Fragmentation Chain Transfer (RAFT)-mediated polymerization was used to synthesise well defined coumarin-containing triazole polymers with moderate PDI values. The polymers were characterised using 1H-NMR and FT-IR. Modelling of the monomer precursor 61 and the monomer 62 at DFT level of approximation provided useful insights into possible conformations adopted by the monomer precursor 61 and the monomer 62. The photophysical properties of the novel monomer and the synthesised polymers were investigated in a polar solvent.

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.

A simple and feasible method of computer colour matching involving fluorescent dyes was developed. An ordinary abridged-spectroreflectometer with polychomatic illumination and a simulated D65 xenon light source was employed for all measurements. In addition to the normal K/S constants for non-fluorescent dyes and the non-fluorescent portion of the fluorescent dye'.. constants responsible for the fluorescent portion were necessary. Two sets of equations to relate the total radiance factors of dyeings with a fluorescent dye and its concentration were developed respectively for self and compound shades where a non-fluorescent dye is admixed. Finding constants responsible for the compound shades required a number of calibration mixture dyeings. Negative K/S constants were found useful when the total radiance factor was above that for the substrate but below one hundred. Three computer programs? s were developed to deal with calibration constants for self and compound shade and also for match prediction. Optimization was used in all cases to minimize errors in total radiance factors or colour differences. Half of the actual dyeingq formulations from the predicted were visually passed by a panel of five dyers. In this study, disperse dyes on polyester were used. Moreover, a commercial matching package was studied using non-fluorescent dyes. The dyeing system affected its accuracy. The polyester/disperse dye system was better than the cotton/reactive dye system. The sample size and luminancefactor of target colours; were also studied. The accuracy was affected slightly by the latter but not the former. / Staff-development Committee and the Institute of Textiles and Clothing of the Hong Kong Polytechnic.

Integral membrane proteins represent nearly 25% of protein encoding genes yet account for less than 2 percent of solved structures in protein databases. This underrepresentation is due to the difficulty in membrane protein isolation with the use of detergents. The difficulty in structural characterization following isolation has been alleviated by use of amphipathic polymers that stabilize proteins by maintaining the native protein environment. Of these amphipathic copolymers, SMA2000—a commercially available styrene-maleic anhydride copolymer with a 2:1 styrene to maleic anhydride ratio—has demonstrated broad utility in the isolation and subsequent characterization of a wide range of integral membrane proteins. However, there are some limitations to the use of SMA2000 that may arise from a lack of control of polymer parameters such as sequence, composition, and dispersity. Methyl substituted stilbene and maleic anhydride copolymers with controlled sequences and compositions demonstrated membrane solubilization and protein isolation activity at levels comparable to SMA2000, with several advantages that include an increase in the useable pH range and the size and homogeneity of polymer-lipid particles. Regrettably, the synthesis of methyl substituted stilbene-maleic anhydride copolymers is not without challenges that would make laboratory scale-up difficult. The use of anisole as a polymerization solvent mitigates gelation during poly((E)-4-methylstilbene-alt-maleic anhydride) synthesis. Additionally, unsymmetrical dimethyl-substituted stilbenes and monosubstituted stilbenes ((E)-4-tert-butylstilbene, (E)-4-methoxystilbene, (E)-2-methoxystilbene, methyl (E)-4-carboxylatestilbene, and (E)-4-trifluoromethylstilbene) copolymerize with maleic anhydride without gelation. Rates of conversion reveal that stilbenes with donor substituents copolymerize with maleic anhydride faster than stilbenes with acceptor substituents. The reversible addition–fragmentation transfer (RAFT) controlled radical polymerization technique afforded poly((E)-4-methylstilbene-alt-maleic anhydride) copolymers with controlled sequence, composition, molecular weight, and dispersity. Amphipathic copolymers with fluorescent activity are also desired for membrane protein solubilization. A series of donor-acceptor di-substituted stilbene and maleic anhydride copolymers with fluorescence activity have been synthesized and characterized. These copolymers possess electron-donating and electron accepting substituents on each phenyl ring of the stilbene monomer. These copolymers exhibit red-shifts that vary in position and width with changes in substituent. / Doctor of Philosophy / Proteins embedded in biological membranes—integral membrane proteins—are valuable drug targets due to their significance in a variety of essential cellular functions such as signaling and transport. Structural and functional characterization of integral membrane proteins is necessary to develop drugs that target them. However, despite their importance, integral membrane proteins remain a challenging target for structural characterization. Integral membrane proteins must be removed or solubilized from the lipid bilayer for structural characterization and the typical detergent extraction process often results in protein destabilization or irreversible protein deactivation. Polyanions have emerged as suitable alternatives to the solubilization of integral membrane proteins. Unlike detergents, polyanions solubilize integral membrane proteins with a small portion of the lipid bilayer intact. In this way, the integral membrane protein is removed from the lipid bilayer, but it maintains its structural and functional integrity allowing for structural and functional characterization. Hydrolyzed poly(styrene-co-maleic anhydride) is the most widely used polyanion for integral membrane protein solubilization, however, the sequences and compositions of poly(styrene-co-maleic anhydride) copolymers are not uniform and these inconsistencies have been shown to effect isolated integral membrane protein yield and purity. Hydrolyzed strictly alternating copolymers of substituted-stilbene and maleic anhydride have uniform sequences and compositions and have demonstrated integral membrane protein extraction activity at levels comparable to or greater than the commonly used hydrolyzed poly(styrene-co-maleic anhydride) copolymers. The work described in this dissertation focuses on optimized syntheses and characterization of several substituted-stilbene and maleic anhydride copolymers for the solubilization of integral membrane proteins. The choice of stilbene substituent is shown to affect poly(substituted-stilbene-alt-maleic anhydride) properties such as solubility and fluorescence activity.