Biophysical and structural characterization of a sequence-diverse set of solute binding proteins for aromatic compounds

Abstract Rhodopseudomonas palustris metabolizes aromatic compounds derived from lignin degradation products and has the potential for bioremediation of xenobiotic compounds. We recently identified four possible solute-binding proteins in R. palustris that demonstrated binding to aromatic lignin monomers. Characterization of these proteins in the absence and presence of the aromatic ligands will provide unprecedented insights into the specificity and mode of aromatic ligand binding in solute-binding proteins. Here, we report the thermodynamic and structural properties of the proteins with aromatic ligands using isothermal titration calorimetry, small/wide angle x-ray scattering, and theoretical predictions. The proteins exhibit high affinity for the aromatic substrates with dissociation constants in the low micromolar to nanomolar range. The global shapes of the proteins are characterized by flexible ellipsoid-like structures with maximum dimensions in the 80–90-A range. The data demonstrate that the global shapes remained unaltered in the presence of the aromatic ligands. However, local structural changes were detected in the presence of some ligands, as judged by the observed features in the wide angle x-ray scattering regime at q ∼0.20–0.40 A−1. The theoretical models confirmed the elongated nature of the proteins and showed that they consist of two domains linked by a hinge. Evaluation of the protein-binding sites showed that the ligands were found in the hinge region and that ligand stabilization was primarily driven by hydrophobic interactions. Taken together, this study shows the capability of identifying solute-binding proteins that interact with lignin degradation products using high throughput genomic and biophysical approaches, which can be extended to other organisms.