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Figure legends
Figure 1. Three-dimensional structure of OspA. Residues substituted to cysteine and with the attached spin-labeled MTSL are depicted as spheres. OspA contains the N-terminal (β1–β7) and C-terminal (β11 to the C-terminus) globular domains, which are connected by a single-layer β-sheet (β8–β10). The image was prepared using Chimera.33 Internal cavities were detected using CASTp server with 1.4 Å radius probe.34
Figure 2. 1H/15N-HSQC spectra of the reduced OspA E128C variant form as pressure was gradually increased from 0.1 to 250 MPa at 313 K (black, 0.1 MPa; blue, 50 MPa; cyan, 100 MPa; green, 150 MPa; purple, 200 MPa; red, 250 MPa). (Inset) Change in chemical shifts of V199 (not visible in the main plot).
Figure 3. Resonance intensities (i.e. peak volumes) of the OspA E128C variant at different pressures and temperature conditions. (A) Resonance intensities at 250 MPa relative to those at 0.1 MPa (313 K) as a function of residue number. (B) Resonance intensities at 318 K relative to those at 303 K (0.1 MPa) as a function of residue number.
Figure 4. Intensity ratios,I para/I dia, for each amide group of the E128C variant for each residue. (A) Pressure dependence of the I para/I dia value at 313 K. (B) Temperature dependence of theI para/I dia value at 0.1 MPa. Secondary structures are indicated at the top of the panels. Error bars for I para/I dia were estimated from the noise considering error propagation, i.e.\(\left(\frac{I_{\text{para}}}{I_{\text{dia}}}\right)=\left(\frac{I_{\text{para}}}{I_{\text{dia}}}\right)\sqrt{\left(\frac{I_{\text{para}}}{I_{\text{para}}}\right)^{2}+\left(\frac{I_{\text{dia}}}{I_{\text{dia}}}\right)^{2}}\).
Figure 5. Pressure and temperature dependence of peak height ratios of a paramagnetic sample (E128C) to those of a diamagnetic sample, i.e.,I para/I dia. (A) Locations of residues 59, 62, 82, 83, and 128 in OspA. (B) Pressure-induced changes in the ratios of residues 59, 82, and 83 at 313 K. (C) Temperature-induced changes in the ratios of residues 59, 62, 82, and 83 at 0.1 MPa. Panel A was prepared using Chimera.33
Figure 6. Pressure- and temperature-dependent effects on selected HSQC cross-peaks. (A) Selected 1H/15N HSQC spectral regions for the reduced MTSL-E128C variant at multiple pressures (black, 0.1 MPa; blue, 50 MPa; cyan, 100 MPa; green, 150 MPa; purple, 200 MPa; red, 250 MPa). (B) Selected1H/15N HSQC spectral regions of the protein at multiple temperatures (black, 303 K; blue, 308 K; green, 313 K; red, 318 K). Assignments for G36 and G106 peaks are depicted at the top of the 0.1 MPa-peak in panel A or 303 K-peak in panel B. The peaks of G36 and G106 are marked with asterisks. (C)I para/I dia values of original and new peaks of residues 36, 37, and 106 at 200 MPa. (D)I para/I dia values of the averaged peaks of residues 36, 37, and 106 at 303 and 318 K.
Figure 7. Frequency ofI para/I dia values calculated from HSQC cross-peaks for the A140C variant. (A) Frequency ofI para/I dia of original cross-peaks at 0.1 MPa and 303 K, corresponding to the native state. (B) Frequency of I para/I dia of new cross-peaks at 250 MPa and 303 K, corresponding to the pressure-stabilized intermediate. (C) Frequency ofI para/I dia of new cross-peaks at 0.1 MPa and 318 K, corresponding to the temperature-stabilized intermediate. Broken lines depict value 1.
Figure 8. I para/I diaprofiles on the basis of MD simulation. (A) MD simulation snapshots of heat unfolding (snapshots after 0.2 ns, 62.7 ns, 73.1 ns, and 80.7 ns from top to bottom, respectively). (B)I para/I dia ratios for the conformational ensembles of different levels of disorder in β9-β11 (black, the folded ensemble (16 snapshots in the MD trajectory of 0.1 ns~1.6 ns); red, the partially disordered ensemble 1 (PDE1, 31 snapshots in the MD trajectory of 60 ns~63 ns); blue, the partially disordered ensemble 2 (PDE2, 121 snapshots in the MD trajectory of 63.1 ns~75.1 ns); green, the partially disordered ensemble 3 (PDE3, 65 snapshots in the MD trajectory of 75.2 ns~81.6 ns)).I para/I dia ratios are calculated from the distance distribution between the Cβof residue 118 (top)/128 (middle)/140 (bottom) (a substitute for the paramagnetic center) and the amide proton of each residue withτ r of 12 ns (see Fig. S14). Panel A was prepared using Chimera.33