H-1, N-15 AND C-13 RESONANCE ASSIGNMENTS, SECONDARY STRUCTURE, AND THE CONFORMATION OF SUBSTRATE IN THE BINARY FOLATE COMPLEX OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE
FALZONE, C. J., CAVANAGH, J., COWART, M., Palmer III, A. G., MATTHEWS, C. R., BENKOVIC, S. J., & WRIGHT, P. E. (1994). H-1, N-15 AND C-13 RESONANCE ASSIGNMENTS, SECONDARY STRUCTURE, AND THE CONFORMATION OF SUBSTRATE IN THE BINARY FOLATE COMPLEX OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE. Journal of Biomolecular NMR, 4(3), 349-366.
By using fully N-15- and N-15/C-13-labeled Escherichia coli dihydrofolate reductase, the sequence-specific H-1 and N-15 NMR assignments were achieved for 95% of the backbone resonances and for 90% of the C-13(alpha) resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected N-15 and C-13 experiments. A smaller but significant subset of side-chain H-1 and C-13 assignments were also determined. In this complex, only one N-15 or C-13 resonance was detected per N-15 or C-13 protein nucleus, which indicated a single conformation. Proton-detected C-13 experiments were also performed with unlabeled DHFR, complexed with C-13-7/C-13-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on H-1 NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667-9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.