Backbone and tryptophan side-chain dynamics of uniformly 15N-labeled Escherichia coli dihydrofolate reductase were determined for the binary folate complex. The 15N T1 and T2 relaxation times and [1H]-15N heteronuclear NOEs were measured for 118 protonated backbone nitrogen atoms. The generalized order parameter (S2), the effective correlation time for internal motions (tau e), and the contribution to spin-spin relaxation through 15N exchange broadening (Rex) were determined for each residue by model-free analysis. Back-calculation of the relaxation rates for each resonance showed that the calculated dynamical parameters accurately predict the experimental data. Diverse dynamical features were evident in the DHFR backbone. Six sites exhibited order parameters significantly below the weighted mean S2 value (for the complex) of 0.81 +/- 0.002: residues G67 and D69 of the adenosine binding domain, and "hinge" residues K38 and V88, exhibited low S2 (0.29 < or = S2 < or = 0.6) and high tau e values (700 ps < or = tau e < or = 2 ns), as did sites within the beta A-alpha B loop and the beta F-beta G loop. Thus, large amplitude backbone motions, on the picosecond and nanosecond time scales, occurred at regions implicated in transition-state stabilization and in ligand-dependent conformational change. Significant Rex values (> or = 1 s-1) were determined for 45% of assigned resonances, many of which arise from residues surrounding the folate binding site. The mean S2 values of the occupied folate binding site and the unoccupied NADPH binding site were similar, indicating the backbone of the latter is at least as conformationally restricted as that of the occupied folate site. We conclude that the observed time-dependent structural fluctuations of the binary complex are in fact associated with catalytic properties of the molecule.