Random Isotopolog Libraries for Protein Perturbation Studies. ¹³C NMR Studies on Lumazine Protein of Photobacterium leiognathi

Lumazine proteins of luminescent bacteria are paralogs of riboflavin synthase which are devoid of catalytic activity but bind the riboflavin synthase substrate, 6,7-dimethyl-8-ribityllumazine, with high affinity and are believed to serve as optical transponders for bioluminescence emission. Lumazine protein of Photobacterium leiognathi was expressed in a recombinant Escherichia coli host and was reconstituted with mixtures (random libraries) of ¹³C-labeled isotopologs of 6,7-dimethyl-8-ribityllumazine or riboflavin that had been prepared by biotransformation of [U−¹³C₆]-, [1-¹³C₁]-, [2-¹³C₁]-, and [3-¹³C₁]glucose. ¹³C NMR analysis of the protein/ligand complexes afforded the assignments of the ¹³C NMR chemical shifts for all carbon atoms of the protein-bound ligands by isotopolog abundance editing. The carbon atoms of the ribityl groups of both ligands studied were shifted up to 6 ppm upon binding to the protein. Chemical shift modulation of the side chain and chromophore carbon atoms due to protein/ligand interaction is discussed on the basis of the sequence similarity between lumazine protein and riboflavin synthase.