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

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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.

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