An analysis of protein-protein interactions: The B1 domain of protein G and Fc fragment of IgG

Protein-protein interactions play a crucial role in many biological processes. Many diseases are caused by aberrant protein-protein interactions and by studying these interactions, effective pharmacological interventions can be developed.; As a model system, the interaction between the B1 domain of protein G and the Fc fragment of IgG was studied. The B1 domain is a small, stable domain that has been characterized structurally both alone and in complex with IgG fragments. The three dimensional structure of the interface between the B1 domain and Fc fragment was used to identify amino acids located at the periphery of the interface that changed their solvent accessible surface area upon complex formation. Environmentally sensitive fluorophores were covalently coupled at these interfacial positions to enable detection of binding between these two proteins.; The energetic consequences of mutating each residue within the B1/Fc interface to alanine was investigated. This alanine scanning mutagenesis identified a four residue hot spot within the interface with a knobs-into-holes binding motif. A single alanine mutation (E27A) completely abolishes complex formation and reduces the affinity by greater than 4000-fold. To further probe the importance of both this crucial residue and the entire hot spot, a combinatorial library was constructed.; The allowable diversity for the four hot spot residues within the interface between the B1 domain of protein G and the Fc fragment of IgG, given the constraint of the residues that surround the hot spot, is very limited and is dictated by the tertiary structure of the B1 domain and the knobs-into-holes binding motif.; These studies lead to the design of novel reagents for the detection of antibodies within complex solutions as well as the identification of B1 domain mutants which both facilitate the single step chromatographic separation of Fc and Fab fragments of IgG and have higher affinities for Fab fragments of IgG. These B1 domain mutants will also facilitate the identification of small molecules capable of modulating binding between the B1 domain and Fc fragment of IgG.