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

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-Å 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 Å(-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.

Environment sensing and response mediated by ABC transporters.

BACKGROUND: Transporter proteins are one of an organism's primary interfaces with the environment. The expressed set of transporters mediates cellular metabolic capabilities and influences signal transduction pathways and regulatory networks. The functional annotation of most transporters is currently limited to general classification into families. The development of capabilities to map ligands with specific transporters would improve our knowledge of the function of these proteins, improve the annotation of related genomes, and facilitate predictions for their role in cellular responses to environmental changes. RESULTS: To improve the utility of the functional annotation for ABC transporters, we expressed and purified the set of solute binding proteins from Rhodopseudomonas palustris and characterized their ligand-binding specificity. Our approach utilized ligand libraries consisting of environmental and cellular metabolic compounds, and fluorescence thermal shift based high throughput ligand binding screens. This process resulted in the identification of specific binding ligands for approximately 64% of the purified and screened proteins. The collection of binding ligands is representative of common functionalities associated with many bacterial organisms as well as specific capabilities linked to the ecological niche occupied by R. palustris. CONCLUSION: The functional screen identified specific ligands that bound to ABC transporter periplasmic binding subunits from R. palustris. These assignments provide unique insight for the metabolic capabilities of this organism and are consistent with the ecological niche of strain isolation. This functional insight can be used to improve the annotation of related organisms and provides a route to evaluate the evolution of this important and diverse group of transporter proteins.

Investigation of the stereoselectivity of an anti-amino acid antibody using molecular modeling and ligand docking.

The structure of the binding site of the stereoselective anti-D-amino acid antibody 67.36 was modeled utilizing web antibody modeling (WAM) and SWISS-MODEL. Although docking experiments performed with an aromatic amino acid as model ligand were unsuccessful with the WAM structure, ligand binding was achieved with the SWISS-MODEL structure. Incorporation of side-chain flexibility within the binding site resulted in a protein structure that stereoselectively binds to the D-enantiomer of the model ligand. In addition to four hydrogen bonds that are formed between amino acid residues in the binding site and the ligand, a number of hydrophobic interactions are involved in the formation of the antibody-ligand complex. The aromatic side chain of the ligand interacts with a tryptophan and a tyrosine residue in the binding site through pi-pi stacking. Fluorescence spectroscopic investigations also suggest the presence of tryptophan residues in the binding site, as ligand binding causes an enhancement of the antibody's intrinsic fluorescence at an emission wavelength of 350 nm. Based on the modeled antibody structure, the L-enantiomer of the model ligand cannot access the binding site due to steric hindrance. Additional docking experiments performed with D-phenylalanine and D-norvaline showed that these ligands are bound to the antibody in a way analogous to the D-enantiomer of the model ligand.