Detection of Open and Closed Conformations of Tryptophan Synthase by 15N-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance of Bound 1-15N-L-Tryptophan

Abstract

1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by tryptophan synthase. 1- 15N-L-Trp was complexed with wild-type tryptophan synthase and β-subunit mutants, βK87T, βD305A, and βE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium α-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1- 15N-L-Trp bound to the β-active site. No 15N-HSQC signal was detected for 1-15N-L-Trp in 10 mM triethanolamine hydrochloride buffer at pH 8. 1-15N-L-Trp in the presence of wild-type tryptophan synthase in the absence or presence of 50 mM sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-L-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium α -glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of βK87T and βE109D mutant Trp synthase with 1-15N-L-Trp showed a similar cross peak either in the presence or absence of disodium α-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the βD305A Trp synthase mutant only showed a 15N-HSQC signal in the presence of disodium α-glycerophosphate. Thus, this mutant Trp synthase favored an open conformation in the absence of disodium α-glycerophosphate but was able to form a closed conformation in the presence of disodium α -glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-L-Trp bound to Trp synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp synthase in the presence of L-Trp with NaCl and/or disodium α-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria.