19F NMR Spectroscopy of [6-19F]Tryptophan-Labeled Escherichia coli Dihydrofolate Reductase: Equilibrium Folding and Ligand Binding Studies

Abstract

Escherichia coli dihydrofolate reductase contains five tryptophan residues distributed throughout its structure. In order to examine the regions of the protein surrounding these tryptophan residues, we have incorporated 6-fluorotryptophan into the protein. To assign the five resonances observed in the 19F NMR spectrum, five site-directed mutants of the enzyme were made, each with one tryptophan replaced by a phenylalanine. The 19F NMR spectra of the apoprotein, two binary complexes (with NADPH or methotrexate), and one ternary complex (with NADPH and methotrexate) were obtained. The chemical shifts of two of the tryptophan resonances (at positions 22 and 74) are particularly sensitive to ligand binding, while the remaining three (at positions 30, 47, and 133) change, but by less. Since several of the tryptophans are distant from the binding site, these results suggest that 19F NMR can detect ligand-induced changes that are propagated throughout the structure. In the apoprotein, the resonances of the tryptophans at positions 22 and 30 are broadened. In the binary complex with NADPH, the resonances of tryptophans 30 and 74 are broadened while that of tryptophan 22 almost disappears. The line broadening of the tryptophan 22 resonance may reflect motion in that part of the protein, since it is near a region that is disordered in the crystal structure of the apoprotein and its NADP+ complex. In contrast, in the ternary complex this region has a defined structure, and all resonances are of equal intensity and line width. The 19F NMR spectra of the apoprotein and the three ligand complexes were also examined as a function of urea concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural differences between apolipoprotein E3 and E4 as measured by 19F NMR

“…Compared to data with other proteins 15,19 and with that of the N-terminal fragment in Figure 1, the spectra suggest that there is considerable conformational heterogeneity in the whole protein presumably induced by the presence of the C-terminal domain as discussed below. This conclusion is strengthened by the observation of a large peak at the resonance of free 5 19 F-tryptophan suggesting that in the whole protein at least three to four of the seven tryptophan residues are solvent exposed and some may be located in disordered regions.…”

“…The appearance of peaks at the chemical shift of free 19 F-tryptophan is typical of a denatured protein. 15,19 Those peaks associated with the N-terminal domain (from À47.5 to À49 ppm) all appear to decrease equally as the protein denatures. Similar denaturation experiments with the C-terminal mutant of apoE3 are shown in Figure 6.…”

“…[12][13][14][15][16][17] The 19 F nucleus is very sensitive to its surroundings and small changes in the structure can be detected by chemical shifts. Additionally, the use of a 19 F cryoprobe allows spectra to be collected at reasonably low protein concentrations and, in general, the 1D spectra are relatively uncomplicated.…”

Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase.

“…E. coli DHFR contains five tryptophan residues distributed throughout its structure. We have previously prepared 6-19F-tryptophan labeled E. coli DHFR, assigned the resonances observed in the 19F spectrum of this protein to individual tryptophans, and studied its behavior at equilibrium in the presence of chemical denaturant (13).In this paper, we monitor the real-time changes in the NMR spectrum of 6-19F-tryptophan labeled E. coli DHFR to study the behavior of side chains during the urea-induced unfolding process. To accomplish this, we have used a stopped-flow device incorporated into the NMR spectrometer (14).…”

“…DHFR used in stoppedflow NMR studies was purified from 500-mi cultures of E. coli strain W3110 trpA33 containing the plasmid pMONDHFR as described (13). This protein was o40% labeled with 6-19F-tryptophan.…”