Photo-CINDP studies of the influence of ligand binding on the surface accessibility of aromatic residues in dihydrofolate reductase

Abstract

The surface accessibility of the histidine, tyrosine and tryptophan residues of Lactobacillus casei dihydrofolate reductase was determined from 360-MHz 1H photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR experiments. In the absence of ligands, 4 (or perhaps 5) of the 7 histidine residues and at least 1 of the 4 tryptophan residues are accessible to a flavine dye molecule. One of the 5 tyrosine residues is also slightly accessible. Of the accessible histidine residues, one becomes inaccessible on the binding of NADP+ and one on the binding of p-aminobenzoyl glutamate. These were assigned to residues which interact directly with these 2 ligands. One histidine residue (probably His-22) shows an increase in accessibility on addition of folate or methotrexate to the enzyme.cntdot.NADP+ complex. In addition, the binding of several ligands, notably trimethoprim, leads to an increase in the accessibility ofa tryptophan residue. This is clear evidence for ligand-induced conformational changes in dihydrofolate reductase and allows identification of some of the residues involved.