This study focuses on the secondary structure of invasion plasmid antigen B (IpaB) of Shigella flexneri and the effects on it by the binding of invasion plasmid gene C (IpgC) along with sites of interaction with IpgC. IpgC is the molecular chaperone of IpaB that is responsible for keeping IpaB from associating with invasion plasmid antigen C (IpaC) or degrading before it is secreted. A library of single tryptophan containing mutants was created by replacing the native tryptophan (Trp) at position 105 with phenylalanine (Phe) and using this as a template to make twelve mutants by inserting Trp at places throughout the length of the protein. Fluorescence spectroscopy utilizing the naturally fluorescent properties of Trp was used to determine areas of interaction with IpgC and the effects of IpgC's binding on IpaB's secondary structure and topology. Invasion and hemolysis assays showed that the native Trp at residue 105 could be replaced with Phe and used as a template to make single insertion mutants. The same assays showed that several of the mutants had no effect on the protein's function while some of the mutants showed no invasiveness or hemolysis but no significant structural changes. Three mutants were determined to be problematic and their use was discontinued. The emission maximum scans showed that many of the mutants were not affected by the binding of IpgC. Two mutants plus the wildtype IpaB displayed significant spectral shifts to the red, indicating that the environment around that Trp became more polar (more hydrophilic) when not bound to IpgC. The thermal unfolding analysis showed that IpaB was more stable when in complex with IpgC than when alone except in one case (for the mutant I553W). The Frster resonance energy transfer (FRET) studies provided information to determine the tertiary structure features of IpaB. These data support the hypothesis that IpaB is a highly structured and complex molecule that is more thermally stable when bound to its chaperone IpgC.Department of Biochemistry and Molecular Biolog
