Structural, biochemical, and computational characterization of beta-lactamase/BLIP interactions

Protein-protein interactions are responsible for many biological processes, and are as diverse as the functions they regulate. However, their varied nature has made it difficult to develop general rules governing the nature of protein-protein interactions, and to quantify the molecular determinants of specificity and affinity. In this thesis work, the structural, biochemical, and computational characterization of beta-lactamase/BLIP interactions is presented, utilizing BLIP's interactions with SHV-1, TEM-1, and KPC-2.;In order to understand the difference in BLIP's affinity for its targets, a key specificity determinant (between SHV-1 and TEM-1) is investigated using structural and mutagenesis methods. The SHV-1 D104E/BLIP co-structure is reported, and mutagenesis of the positions involved in an interfacial salt bridge demonstrates that the presence of a single destabilizing interaction in the SHV-1/BLIP interface is primarily responsible for the reduced affinity (compared to TEM-1). Structural and biochemical responsible for the reduced affinity (compared to TEM-1). Structural and biochemical characterization of two BLIP mutants (BLIP E73M and BLIP E73M/S130K/S146M) in complex with SHV-1 further support the significance of this area to affinity. Extending BLIP's repertoire of targets, the crystallographic structure of the unreported KPC-2/BLIP complex is presented.;Towards the goal of quantifying the physical forces responsible for affinity in protein-protein interactions, the EGAD Library is established as a suitable protein design algorithm. EGAD performs similarly to other published methods on a benchmark predicting the effects of mutation on protein-protein binding energy. With knowledge gained from the presented SHV-1/BLIP mutant structures, the computational model was adjusted in attempts to more correctly capture sidechain conformation. Computational alanine scanning on the KPC-2/BLIP interface identifies many previously known BLIP hot spots to also be important in the KPC-2 interface.;Finally, in order to facilitate higher-throughput mutagenesis studies, a novel method for determining binding affinities is described using the TEM-1/BLIP system.