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This study focuses on the modulation of protein aggregation and immunogenicity. As a starting point for investigating long-range interactions within a non-native protein, the effects of perturbing denatured protein states on their aggregation, including the formation of amyloid fibrils, were evaluated. The effects of adducts, sugar modifications, and stabilization on protein aggregation were then examined. We also investigated how protein immunogenicity was affected by enhancing protein conformational stability and other factors.

DnaA protein binds specifically to a group of binding sites called DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. DnaA domain IV comprises 94 amino acid residues and is required for DNA binding. At first, the backbone assignments of DnaA domain IV in the complexes were determined using several nuclear magnetic resonance (NMR) spectra. Using NMR analysis, we investigated the interaction between DnaA domain IV and DnaA box R1. The 1H-15N HSQC spectrum of DnaA domain IV showed prominent chemical shift perturbations on six residues (Arg399, Ala404, Leu422, Asp433, Thr435 and Thr436). Through homology modeling, we located all of these residues on one side of the surface of the DnaA domain IV molecule. Moreover, we confirmed that these residues in DnaA domain IV bind to DnaA box R1 by mutation analysis. Finally, we compared the chemical shift perturbation of the 1H-15N HSQC spectrum in the presence of the DnaA box with that in the presence of a non-specific oligonucleotide that has a reduced affinity for DnaA, and the results suggested that Leu422 imparts specificity in binding with DnaA box R1.