SUPPLEMENTARY INFORMATION

Transcription

1 5 N a b H c (kda) Precipitate before NMR expt. Supernatant before NMR expt. Precipitate after hrs NMR expt. Supernatant after hrs NMR expt. Supernatant after hrs NMR expt. (x3) N d e H Supplementary Figure E. coli cells expressing TTHA78 under NMR measurement conditions. 2D H- 5 N HSQC spectra of a TTHA78 in-cell NMR sample: a, immediately after sample preparation; b, the lysate of the harvested cells after hours NMR measurement. c, SDS-PAGE with Coomassie staining performed on in-cell NMR samples demonstrating that the proteins providing the NMR spectra in Fig. b and c (corresponding to lanes and 3, respectively, in supplementary Fig. c) are indeed inside the living cells and the contribution of extracellular protein to the observed signals is negligible. 2D H- 5 N HSQC spectra of the spheroplasts (d) and periplasmic extract (e), which were fractionated from TTHA78-expressing 5 N-labelled E. coli cells by Lysozyme-EDTA treatment, indicating that overexpressed TTHA78 is in cytoplasm. The spheroplasts were suspended in an isotonic buffer. The measurement time was increased twentyfold for the periplasmic extract sample in consideration of the dilution during the preparation of the periplasmic extract. The cytoplasmic localisation of TTHA78 was also supported by predictions from its amino acid sequence by PSORTb v.2.0 ( and SignalP 3.0 (

2 a 5 N b C 3 45 T0 0 HNCA HN(CO)CA E7 T45 E29 G44 A K37 L35 L5 K23E39 K49 L22 T9 V52 E58 V55 E3 E3 Q53 V5 V8 L5 K M9 S34 K20 A2 Y0 A54 E57 E5 V3 V28 A40 D47 G59 A50 L4 L4 C4 K3 M V42 V4 A7 A4 K5 E32 A2 V25 V33 G8 L2 E43 V G27 G H L4 V42 E43 G44 T45 A4 c H HBHA(CBCACO)NH H(CCCO)NH L4 V42 E43 G44 T45 A4 A40H β V42H γ T45H γ 50 2 L4H β E43H β E43H γ A40H α G44H α T45H α L4H α V42H α G44H α E43H α d H H Δave (Hz) Residues Supplementary Figure 2 Backbone and side-chain resonance assignments of TTHA78 in living E. coli cells. a, 2D H- 5 N HSQC spectrum of TTHA78 in living E. coli cells. Cross peaks are labelled with their corresponding backbone assignments. b, Overlaid H N - 3 C α cross-sections of the 3D HNCA (black) and the HN(CO)CA (red) spectra 2

3 corresponding to the 5 N frequencies of residues from Leu4 to Ala4. Sequential connectivities are represented by dashed red lines. c, Overlaid H N - H cross-sections of the 3D HBHA(CBCACO)NH (black) and the H(CCCO)NH (green) spectra corresponding to the same residues presented in b. d, A plot of chemical shift differences of backbone H N and 5 N nuclei between in-cell and in vitro conditions. The weighted shift difference Δ ave for each amino acid residue was calculated as [(Δ H N ) 2 + (Δ 5 N) 2 ] /2 where Δ H N and Δ 5 N are the chemical shift differences (Hz; ppm corresponds to 00.3 Hz for H and 0.8 Hz for 5 N) between the two conditions. The residues in which H- 5 N correlation cross peaks were not observed either in cell or in vitro are represented in yellow. The positions of two proline residues are shown in grey. 3

4 8 a b c 20 3 C d H H H C A7β A2β.2 A54β V5γ A50β Vγ Vγ Vγ' Vγ Aβ V4γ' Vγ A4β V8γ' V4γ Vγ V33γ Vγ V28γ A40β L5δ V25γ' L35δ A2β Vγ V3γ V8γ L5δ Lδ V5γ' Vγ Lδ V28γ' L5δ' Lδ H L22δ' Lδ L22δ L5δ' L35δ' 0.2 e 3 C L5H N L5H N E32H N E32H N E3H N L35H N A50 Cβ H A50 Hβ C V3 Cγ H H V3 Hγ 3 C L35 Cδ L35 Cδ' H L35 Hδ L35 Hγ Hβ Supplementary Figure 3 Assignments of side chain methyl groups of TTHA78 in living E. coli cells. 2D H- 3 C HMQC spectra of TTHA78 in-cell NMR samples with three different methyl-selective labelling patterns, Ala/Leu/Val (a), Ala/Val (b) and Leu/Val (c), which were used for amino acid classification of methyl H- 3 C correlation cross peaks. d, 2D H- 3 C HMQC spectrum of TTHA78 in living E. coli cells. Cross peaks are labelled with their corresponding assignments. e, Overlaid H N - 3 C α or H N - H cross-sections of the 3D CBCA(CO)NH (black), 3D HBHA(CBCACO)NH (black), 3D (H)CC(CO)NH (red), 3D H(CCCO)NH (red) and 3D 5 N-separated NOESY-HSQC (blue) spectra used for the assignments of side-chain methyl groups of A50 C β, V3 C γ and L35 C δ /C δ. 4

5 C H 3 2 Supplementary Figure 4 Background H- 3 C correlation cross peaks originating from uniform 3 C-labelling. Overlay of the H- 3 C HSQC spectra of purified TTHA78 (black) and E. coli cells expressing TTHA78 (red). 5

6 Table NMR structure statistics for TTHA78 a Quantity in-cell in-cell w/o ALV b in vitro Short/medium/long-range distance restraints * 79/24/89 54/8/24 770/34/578 Restrained hydrogen bonds Dihedral angle restraints Maximal distance restraint violation (Å) 0.3 ± ± ± 0.0 Maximal dihedral angle restraint violation (º) 2.2 ± ± 0.7 Deviations from idealized geometry: Bond lengths (Å) ± ± ± Bond angles (º).75 ± ± ± 0.04 AMBER energy (kcal/mol) -249 ± ± ± 72 AMBER van der Waals energy (kcal/mol) -2 ± 3-34 ± ± Ramachandran plot statistics (%) 92/7//0 90/9//0 93/7/0/0 Backbone RMSD (Å) 0.9 ± ± ± 0.04 All heavy atom RMSD, Å.53 ± ± ± 0.05 Backbone RMSD to the in vitro structure (Å) c All heavy atom RMSD to the in vitro structure (Å) d.87.2 a Where applicable, the average value and the standard deviation over the 20 energyrefined conformers that represent the NMR structure are given. b Statistics for TTHA78 calculated without NOE-derived distance restraints involving methyl groups obtained in methyl-selectively protonated in-cell NMR samples. c Backbone RMSD of the mean structure of the ensemble to the in vitro mean structure. d All heavy atom RMSD of the mean structure of the ensemble to the in vitro mean structure.

7 a 4 E7 E29 T45 4 E7 E29 T45 G44 A K37 G44 A K37 K49 K23 L5 E39 K49 E39 S L5 K23 V55 L35 T9 L35 V55 8 E3 S4 E58 8 E3 T9 E58 V52 V52 E3 Q53 E3 Q53 V5 20 V8 L5 20 V8 L5 5 N K 5 Y0 N K K20 Y0 M9 S34 A2 22 K20 22 M9 S34 E57 E57 A54 E5 V5 A54 A2 V3 V28 V3 M A 24 A40 D47 G59 G59 V42 M 24 A50 A40 D47 E5 V28 A50 L4 V42 A7 2 L4 K3 L4 A4 L4 K3 A4 K5 V4 2 A7 A4 E32 K5 V4 A2 A2 E32 28 V33 G8 V25 28 V33 G8 V25 L2 V G38 L2 V G38 E43 G27 E43 G H H c 200 L22 b d 200 L Δave (Hz) 00 Δave (Hz) Residues Residues Supplementary Figure 5 Backbone resonance assignments of two TTHA78 mutants, CS/C4S and CA/C4A, in living E. coli cells and in vitro. a, Overlay of the 2D H- 5 N HSQC spectra of TTHA78(CS/C4S) mutant in living E. coli cells (red) and in vitro (black). b, Overlay of the 2D H- 5 N HSQC spectra of TTHA78(CA/C4A) mutant in living E. coli cells (red) and in vitro (black). For both panels a and b, cross peaks are labelled with their corresponding backbone assignments. For both mutants, all backbone resonances of the non-n-terminal and non-proline residues were assigned except for Thr0, Asn2 and His3. Plots of the chemical shift differences of backbone H N and 5 N nuclei of the CS/C4S mutant (c) and the CA/C4A mutant (d) between incell and in vitro conditions. The shift difference Δ ave for each amino acid residue was calculated as in supplementary Fig. 2d. The residues for which H- 5 N correlation cross peaks were not observed either in cell or in vitro are represented in yellow. The positions of two proline residues were shown in grey. 7

8 5 N 22 a b c 4 T C V5 M C4 28 A H H H Supplementary Figure In vitro characterisation of the metal-binding activity of two TTHA78 mutants, CS/C4S and CA/C4A. 2D H- 5 N HSQC spectra of wild type TTHA78 (a), CS/C4S (b) and CA/C4A (c). For each panel, two spectra measured in M9 medium (red) and in M9 medium supplemented with an excess of a metal salts solution (ZnSO 4, MnSO 4 and CuSO 4 ) (black) are overlaid. The final concentrations of these three metal ions were 200 μm, 50 μm and 35 μm, respectively, which are 50 times higher than the concentrations used to supplement the in cell growth in M9 medium. Upon the addition of the metal mixture to wild type TTHA78, significant line broadening and/or chemical shift changes were found for residues distributed around the putative metalbinding loop (indicated in a), while no significant changes were found for the CS/C4S or CA/C4A mutants, suggesting that these two mutants lack metal-binding activity. 8

9 5 N 22 a b c 4 T V5 M9 24 C H H H Supplementary Figure 7 Characterisation of the metal-binding activity of two TTHA78 mutants, CS/C4S and CA/C4A in E. coli cells. 2D H- 5 N HSQC spectra of wild type TTHA78 (a), CS/C4S (b) and CA/C4A (c) measured in living E. coli cells. Each panel shows two spectra measured in M9 medium (red) and in M9 medium supplemented by an excess of a metal salts solution (ZnSO 4, MnSO 4 and CuSO 4 ) (black) overlaid. The final concentrations of the three metal ions were 200 μm, 50 μm and 35 μm, respectively. The metal mixture was added into the E. coli culture an hour before the cells were harvested. For wild type TTHA78 in E. coli cells in the presence of excess Zn 2+, Mn 2+ and Cu 2+ ions in the medium, additional line broadening and chemical shift changes similar to those seen in the in vitro experiments (supplementary Fig. a) were observed while no significant change was found for either mutant. 9

10 Supplementary Figure 8 The contribution of long-range NOEs involving methyl groups to the structure calculation of TTHA78 in living E. coli cells. a, A superposition of the 20 final structures of TTHA78 in living E. coli cells, showing the backbone (N, C α, C ) atoms. b, A superposition of the 20 final structures of TTHA78 in living E. coli cells calculated without distance restraints derived from NOEs involving methyl groups obtained in methyl-selectively protonated in-cell NMR samples. 0

11 E32 a b E32 c E32 4 d e f 3 hr hr 0.5 hr N g H H ( ppm) H E32 V33 S34 E39 3 hr hr 0.5 hr 3 hr hr 0.5 hr 3 hr hr 0.5 hr 3 hr hr 0.5 hr V3H γ Hγ V33Hγ V33Hγ A40H β K37H β 2 H β Hγ 2 H β 2 V33H β 2 H β H β' Hγ G38Hα G38Hα 4 H 5 Hα V42H α V3H α 4 H 5 E32H α 4 H 5 H β V33H α H α A40Hα 4 H 5 H α S34H β K37H α G38H N S34HN 9 L4H N H H H H Supplementary Figure 9 3D 5 N-separated NOESY-HSQC spectra acquired on TTHA78 in-cell NMR samples with various protein expression levels. The concentration of TTHA78 in in-cell NMR samples collected after 3 hours incubation following induction of protein expression was estimated to be 3-4 mm by SDS-PAGE. 2D H- 5 N HSQC spectra are shown for the in-cell NMR samples with three different incubation times, 3 hours (d), hour (e) and 30 minutes (f) prior to cell harvest. D cross

12 sections taken at the position indicated by the dotted lines are shown above the corresponding 2D spectra (a, b and c, respectively). From the cross peak intensities, the concentrations of TTHA78 in the in-cell NMR samples collected after hour or 30 minutes were estimated to be.2-. and mm, respectively. g, H N - H crosssections corresponding to the 5 N frequencies of residues, Glu32, Val33, Ser34 and Glu39 extracted from the 3D 5 N-separated NOESY-HSQC spectra of TTHA78 in-cell NMR samples with incubation times of 3 hours, hour and 30 minutes prior to cell harvest. All three 3D 5 N-NOESY spectra were measured with essentially identical parameters, and the spectrum with 3 hours incubation was analysed to obtain NOE-derived distance restraints for structure calculations. The cross peaks due to inter-residue and intra-residue NOEs are indicated annotated in red on the spectrum with 3 hours incubation. Intraresidue NOEs are indicated by blue boxes and annotated. Even for the samples with hour and 30 minutes incubation time, we could identify 74% (34) and % (299) of all cross peaks (487) observed for the sample with 3 hours incubation time and used for the structure calculation. 2

13 a b c N T5 Q49 E7 L48 E E45 A4 T44 L4 E47 Q C T44 E45 A4 E47 L48 Q H 5 L4 T5 E E7 Q H HNCA HN(CO)CA H H Supplementary Figure 0 In-cell NMR spectra of rat calmodulin in E. coli cells. a, 2D H- 5 N HSQC spectrum of rat calmodulin in E. coli JM09 (DE3) cells. The concentration of calmodulin in in-cell NMR samples was estimated to be.0-.5 mm by SDS-PAGE. Note that H- 5 N HSQC spectra with equivalent quality were measured when using HMS74(DE3) as host E. coli cells, while H- 5 N correlation cross peaks were extremely broadened when using BL2(DE3) as host cells. b, Overlaid H N - 3 C α crosssections of the 3D HNCA (black) and the HN(CO)CA (red) spectra corresponding to the 5 N frequencies of residues from Thr44 to Gln49. Sequential connectivities are represented by dashed red lines. The positions of the cross peaks due to these residues in 2D H- 5 N-HSQC are indicated in panel a. c, H N - H cross-sections corresponding to the 5 N frequencies of residues from Leu4 to Gln8 extracted from the 3D 5 N-separated NOESY-HSQC spectrum. Sequential connectivities of H N -H N NOEs are indicated as dashed red lines. The positions of the cross peaks due to residues, Leu4 to Gln8 are also indicated in panel a. 3

14 4 8 5 N a c K5 K23/ * K37/K K20/K49 K3 K5 K3 K37 K49 K23 K20 K * N b d K5 K3 K5 K3 K20 K K49 K23 K37 K37 K49 K23 K20 K H H 7 Intensity Intensity.0 K3 K5 K20/K K37/K Time (ms) e g C-term. K3 K5 K20/K49 K37/K Time (ms) K49 i N-term. K3 K5 K Intensity Intensity Time (ms) f h Time (ms) K23 K20 K3 K5 K20 K23 K37 K49 K K3 K5 K20 K23 K37 K49 K K37 Supplementary Figure Longitudinal (T ) and transverse (T 2 ) 5 N relaxation data of TTHA78 in living E. coli cells. 5 N relaxation data in living E. coli cells and in vitro were obtained by measuring D 5 N-edited 5 N T or T 2 relaxation experiments with various relaxation delays on samples selectively labelled with 5 N-lysine. Each relaxation experiment was repeated 4-5 times for statistical analysis. The 2D H- 5 N HSQC spectrum (c) and its D projection (a) of lysine selectively 5 N-labelled TTHA78 in living E. coli cells are shown. Corresponding 2D and D spectra measured in vitro are shown in d and b, respectively. 5 N T and T 2 data for the backbone amide 5 N nuclei of lysine residues of TTHA78 in E. coli cells (e and g) and in vitro (f and h) are displayed with their singleexponential least-squares best-fit curves. Error bars, if not shown, lie within the size of symbols used to indicate the data points. T values were obtained by using relaxation 4

15 delays of 5, 55, 05, 55, 255, 405, 505, 755, 005, 205 and 505 ms. T 2 values were obtained using 5 relaxation delays (4.4, 28.8, 43.2, 72.0 and 00.8 ms) for in-cell samples and relaxation delays (4.4, 43.2, 72.0, 5.2, 72.8 and ms) for in vitro samples. In the analysis of in-cell samples, data for Lys23 were excluded since the amide resonance is overlapped in the acquisition dimension with a sharp background signal (represented with in panel c). The spatial distribution of the 9 lysine residues in TTHA78 is shown in panel i. 5

16 Table 2 5 N T and T 2 relaxation times for backbone 5 N nuclei of lysine residues of TTHA78 in E. coli cells and in vitro, at a spectrometer frequency of 00 MHz and a temperature of 30 K. Resdue Relaxation times (mean and s.d.) T (ms), in-cell T (ms), in vitro T 2 (ms), in-cell T 2 (ms), in vitro Lys3 835 ± 5 4 ± ± ± 2 Lys5 77 ± 4 43 ± ± ± Lys20 43 ± 3 74 ± Lys20/Lys ± ±.2 Lys ± 84 ± Lys24 73 ± ± ± ± Lys ± ± ± ± Lys ± 5 9 ± 2 Lys37/Lys 94 ± ± 2. Lys ± 90 ± Lys 53 ± ± 2