A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C(MDCC) DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MD() binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethy1]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067 mu M(-1) s(-1)), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131C(MDCC) DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131C(MDCC) DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis. (C) 2011 Elsevier Inc. All rights reserved.