Closed Form of Liganded Glutamine-Binding Protein by Rotational-Echo Double-Resonance NMR † Christopher A. Klug, ‡,§ Kenzabu Tasaki, ‡,| Nico Tjandra, ⊥,@ Chien Ho, ⊥ and Jacob Schaefer* ,‡ Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130, and Department of Biological Sciences, Carnegie Mellon UniVersity, Pittsburgh, PennsylVania 15213 ReceiVed March 4, 1997; ReVised Manuscript ReceiVed May 23, 1997 X ABSTRACT: Rotational-echo double-resonance NMR has been used to determine internuclear distances in the complex of glutamine-binding protein and its ligand, L-glutamine. The distances between the ligand and Tyr185 are consistent with the results of molecular dynamics simulations constrained by three REDOR- determined distances to His156. This model is also consistent with six other REDOR-determined internuclear distances, most of which agree with values from the first report of an X-ray structure of the complex of glutamine-binding protein and L-glutamine. We recently reported the determination of several inter- nuclear distances in the complex between glutamine-binding protein (GlnBP) 1 and its ligand, L-glutamine (Hing et al., 1994), using stable-isotope labeling and rotational-echo double-resonance (REDOR) NMR. GlnBP is a 25 kDa protein that is an essential component of the glutamine transport system in Escherichia coli (Ames, 1986). The REDOR-determined distances were also used as constraints in molecular dynamics calculations which led to a proposed structure for the complex. The modeling was limited by the fact that (i) REDOR distances were measured from the ligand to residues in only one of the two GlnBP domains and (ii) the simulations were not sufficiently long to observe a stable closed structure. In this paper, we describe a new type of REDOR measurement which includes the determinations of distances between L-glutamine and residues in both domains. We also compare these distances to those from an extended molecular dynamics simulation, as well as to distances from the first report of an X-ray crystallograpic analysis of the complex (Hsiao et al., 1996). MATERIALS AND METHODS Solid-State NMR Samples and Spectrometer. Isotopically labeled protein was prepared following the procedure described previously by Hing et al. (1994). GLnBP labeled by [ring-4- 13 C]Trp was complexed to L-[amine- 15 N]glutamine in a solution that was 2.7 mg/ml protein, 1% poly(ethylene glycol) 8000, 20 mM sucrose, 2 mM 4-morpholinepropane- sulfonic acid, and 1 mM dithiothreitol (Studelska et al., 1996). The solution containing the ternary complex was frozen at -20 °C and then cooled with liquid nitrogen before lyophilization. Two other complexes were prepared that contained buffer but no cryo- or lyoprotectants. [m- 19 F]Tyr- [uniform- 15 N]GlnBP was complexed as described before (Hing et al., 1994) to (i) L-[5- 13 C]glutamine and (ii) unlabeled L-glutamine. Powdered, lyophilized protein complex (100- 200 mg) was packed into 7.5 mm outside diameter zirconia rotors fitted with Kel-F spacers and drive cap. Cross- polarization, magic-angle spinning spectra were obtained at 4.7 T using a four-channel probe with a single 9 mm diameter solenoidal coil which permits 1 H, 19 F, 13 C, and 15 N detection or dephasing (at 200, 188, 50, and 20 MHz, respectively). Fluorine incorporation into GlnBP was measured by direct 19 F NMR detection, calibrated by comparisons to spectra of materials with known fluorine content. REDOR experiments began after a 2.0 ms matched spin-lock cross-polarization transfer from protons at 50 kHz, followed by proton decoupling at 100 kHz. The sequence repetition time for most experiments was 2 s. There was no indication of large- amplitude molecular motion either from slow cross-polariza- tion transfer rates or from unusually fast spin-lattice relaxation rates. The magic-angle stators were obtained from Chemagnetics (Fort Collins, CO). A controlled spinning speed of 5000 Hz was used for REDOR experiments. REDOR. REDOR provides a direct measure of hetero- nuclear dipolar coupling between isolated pairs of labeled nuclei (Gullion & Schaefer, 1989). In a solid with an I-S- labeled spin pair, for example, the S-spin rotational echoes that form each rotor period following a proton to S-spin cross-polarization transfer can be prevented from reaching full intensity by insertion of two I-spin π pulses per rotor cycle, one in the middle of the rotor period and the other at the completion of the rotor period (for the GlnBP experiments discussed here, I is either 19 F or 15 N and S is 13 C). Both I- and S-spin pulses were applied using an XY8 phase-cycling scheme to suppress offset effects and compensate for pulse imperfections (Gullion et al., 1990). The REDOR difference (the difference between an S-spin NMR spectrum obtained under these conditions and one obtained with no I-spin π pulses) has a strong dependence on the dipolar coupling and hence the internuclear distance. Measurements of carbon- † This work was supported by NIH Grants GM-26874 and HL-24525 (C.H.) and GM-51554 (J.S.). ‡ Washington University. § Present address: Department of Chemical Engineering, Stanford University, Stanford, CA 94305. | Present address: Mitsubishi Chemical America, Inc., San Jose, CA 95134. ⊥ Carnegie Mellon University. @ Present address: Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, MD 20892. X Abstract published in AdVance ACS Abstracts, July 1, 1997. 1 Abbreviations: GlnBP, glutamine-binding protein; REDOR, rota- tional-echo double resonance; NMR, nuclear magnetic resonance. 9405 Biochemistry 1997, 36, 9405-9408 S0006-2960(97)00501-1 CCC: $14.00 © 1997 American Chemical Society