Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

Abstract

Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of atleast 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins’ functions were obtained from their interactions with each of a panel of ligands.

Fig. 1

Nucleotide ligand interaction assay using Mtb recombinant proteins by dye-ligand affinity chromatography. The dye-ligand affinity chromatography results from SDS-PAGE show binding of eight target proteins to Cibacron Blue F3GA and subsequent selective elution in response to exposure to a sequence of eight different nucleotide ligands (NAD, NADH, NADP, NADPH, AMP, ADP, ATP, and GTP)

Fig. 2

Ligand-enhanced crystallization of Mtb recombinant proteins. Proteins were crystallized in the absence (Native, left) or presence (right) of various nucleotide ligands, as indicated. Ligand selection was based on the results of the ligand interaction assay (Fig. 1 and Table 1). Enhanced crystallization of protein–ligand co–crystals, relative to native protein alone, was observed for all eight proteins, and allowed for structural resolution of four proteins (resolution of the collected diffraction data is indicated in parentheses)