Environmentally Ultrasensitive Fluorine Probe to Resolve Protein Conformational Ensembles by 19F NMR and Cryo-EM

Abstract

Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by ¹⁹F NMR. We describe a novel monofluoroethyl ¹⁹F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) ¹⁹F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, ¹⁹F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.

Figure 1

Synthesis of the mFE probes and site-specific protein labeling. Synthesis of deuterated and protonated mFE probes (a) TsSCD₂CD₂F and (b) TsSCH₂CH₂F. (c) Cysteine-specific labeling. (d) Structure of the Glt_Ph trimer with scaffold domains colored tan and the transport domains colored blue. Colored spheres show the sites of single-cysteine mutations A380C (cyan), A381C (magenta), and M385C (yellow). Dashed line indicates the distance between the ¹⁹F labels. (e) Elevator transition of the Glt_Ph transport domain from the OFS (left) to IFS (right). Single protomers are shown in the membrane plane, represented by the dotted lines. The bound substrate aspartate is shown as lime spheres. ACN: acetonitrile; DAST: diethylaminosulfur trifluoride; DCM: dichloromethane; X: hydrogen or deuterium; and Ts: toluenesulfonyl.

Figure 2

mFE^D-labeled Glt_Ph variants show wide chemical shift dispersion. ¹⁹F NMR spectra of (a, b) Glt_Ph-M385C and (c, d) Glt_Ph-A380C labeled with (a, c) tFE and (b, d) mFE^D. From top to bottom, (a, b) WT and RSMR mutants in 100 mM Na⁺ and 2 mM aspartate (Asp) and the RSMR mutant in 100 mM Na⁺ and 0.6 mM TFB-TBOA, and (c, d) WT in 400 mM Na⁺ and 100 mM Na⁺ and 2 mM aspartate. All spectra were recorded at 25 °C. Raw data are black, fitted spectra are pink, and deconvoluted Lorentzian peaks are blue. The asterisk denotes a signal from the CF₃ group of TFB-TBOA. S1–S5 denote resolved resonances of Glt_Ph-M385C-mFE^D. Δδ is the largest chemical shift difference observed for the labeling site. All ¹⁹F NMR spectra here and elsewhere were recorded at least twice on independently prepared protein samples, producing similar results.

Figure 3

Identification and structural elucidation of new transporter conformations. (a) ¹⁹F NMR spectra of WT-M385C-mFE^D (upper panel) and RSMR-M385C-mFE^D (bottom panel) in the presence of 100 mM NaCl and 2 mM TBOA. The resonances occurring with similar chemical shifts to those in Figure 2 are labeled S2, S3, and S4. (b) Cryo-EM density maps of TBOA-bound RSMR Glt_Ph mutant protomers in the IFS (left) and OFS (right) structural classes. The corresponding populations are below the maps.

Figure 4

Assignment of IFS and OFS conformations based on solvent PRE effects. ¹⁹F NMR spectra (left) were recorded, and T₁ relaxation times (right) were measured in 300 mM NaCl and 2 mM Asp at 15 °C. Relaxation data were fitted to monoexponential functions (solid lines). The fitted T₁ values are (a) 1.36 ± 0.20, 1.41 ± 0.02, 1.26 ± 0.33, 1.56 ± 0.16, and 1.26 ± 0.09 s for S2, S3, S4, S5, and S6, respectively, in the absence of Gd-DPTA–BMA and (b) 1.15 ± 0.20, 1.04 ± 0.08, 0.39 ± 0.08, 0.42 ± 0.06, and 1.01 ± 0.06 s in the presence of 20 mM Gd-DPTA–BMA. The error bars, estimated as described in the Materials and Methods Section, are too small to see.

Figure 5

State populations observed in 3D classifications of protomers in cryo-EM parallel NMR measurements. (a) NMR spectra of RSMR-M385C-mFE^D were recorded in the presence of 500 mM NaCl and 2 mM Asp at 4 (top), 15 (middle), and 30 °C (bottom). (b) Representative maps of the four structural classes identified during cryo-EM particle 3D classifications of RSMR-M385C-mFE^D prepared at 4 °C. (c) Populations of protomers’ 3D classes in samples preincubated at 4, 15, and 30 °C (left). Open circles are results obtained using different tau values and numbers of classes during 3D classifications in RELION (see the Materials and Methods Section for details). The state populations measured in ¹⁹F NMR experiments at the same temperatures are shown in the right panel. Errors are from multiple-peak deconvolutions of spectra in OriginPro 2019.