Polydiacetylene-based colorimetric biosensors and structural and kinetic studies of catalytic antibody 7G12: A ferrochelatase mimic

By exploiting the unique properties of diacetylenic lipids, polydiacetylene-based colorimetric sensors that mimic natural cell membranes can be created. To date, all polydiacetylene-based colorimetric sensors have utilized polydiacetylenic matrices made exclusively from single-chain diacetylenic fatty acids. This thesis presents a polydiacetylene-based colorimetric sensor that introduces the use of a novel di-chain diacetylenic phospholipid, 1,2-bis(10,12-tricosadiynoyl)- sn-phosphocholine, to the repertoire of diacetylenic lipids used in sensor design. To demonstrate the usefulness of this lipid, a sensor was developed to detect phospholipase A₂, an enzyme which binds to and cleaves cell membrane phospholipids in their natural environment. In addition to allowing polydiacetylene-based colorimetric sensors to better mimic the lipid composition of natural cell membranes, 1,2-bis(10,12-tricosadiynoyl)- sn-phosphocholine decreases lipid headgroup packing within the polydiacetylene matrix which may prove to be advantageous in developing sensors less susceptible to false positives. The understanding gained from the development of this phospholipase A₂ sensor could be applied towards the development of sensors designed to detect a variety of lipase containing pathogenic agents and enzyme inhibitor drug discovery.; In addition to the aforementioned sensor studies, kinetic and structural studies of catalytic antibody 7G12, a ferrochelatase mimic, were also performed. Previously, the structure of the affinity-matured 7G12 antibody bound to hapten N-methylmesoporphyrin was determined to a resolution of 2.4 Å. This thesis presents the crystal structures of both the unliganded (1.6 Å) and liganded (1.8 Å), with regard to hapten, forms of the germline precursor 7G12 antibody along with the apo (2.2 Å) and substrate-bound Michaelis-complex (2.6 Å) forms of the affinity-matured 7G12 antibody. Kinetic studies of the germline and affinity-matured 7G12 antibodies support a fundamental hypothesis of catalytic antibody efficacy, that as an antibody's binding affinity towards hapten increases so does its rate of substrate catalysis. Structural studies of the germline and affinity-matured 7G12 antibodies bound and unbound to hapten support the hypothesis that enzymes evolve from an induced fit mechanism towards a lock-and-key mechanism of substrate binding and catalysis. Lastly, structural studies of the Michaelis-complex reveals that antibody 7G12 induces strain within the substrate mesoporphyrin, providing unequivocal structural evidence in support of Haldane's theory of strain in enzyme catalysis.