nature template - pc word 97 · 2020-03-06 · 2d 1h-15n hsqc spectra of a ttha1718 in-cell nmr...

15N

(ppm

)

1H (ppm)10 9 8 7 6

114116118120122124126128

114116118120122124126128

a

b

2128

4157

110195

(kDa)

15

6.5 1 2 3 4 5Pr

ecip

itate

bef

ore

NM

R e

xpt.

Supe

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ant b

efor

e N

MR

exp

t.

Prec

ipita

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fter 6

hrs

NM

R e

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Supe

rnat

ant a

fter 6

hrs

NM

R e

xpt.

Supe

rnat

ant a

fter 6

hrs

NM

R e

xpt.

(x3)

c

15N

(ppm

)

1H (ppm)10 9 8 7 6

114116118120122124126128

114116118120122124126128

d

e

Supplementary Figure 1 | E. coli cells expressing TTHA1718 under NMR measurement conditions. 2D 1H-15N HSQC spectra of a TTHA1718 in-cell NMR sample: a, immediately after sample preparation; b, the lysate of the harvested cells after 6 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. 1b and 1c (corresponding to lanes 1 and 3, respectively, in supplementary Fig. 1c) are indeed inside the living cells and the contribution of extracellular protein to the observed signals is negligible. 2D 1H-15N HSQC spectra of the spheroplasts (d) and periplasmic extract (e), which were fractionated from TTHA1718-expressing 15N-labelled E. coli cells by Lysozyme-EDTA treatment, indicating that overexpressed TTHA1718 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 TTHA1718 was also supported by predictions from its amino acid sequence by PSORTb v.2.0 (http://www.psort.org/psortb/) and SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/).

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1H (ppm)8.9 9.0 8.8 8.8 8.3 7.1

1

2

3

4

5

45

50

55

60

65

8.9 9.0 8.8 8.8 8.3 7.1

L41 V42 E43 G44 T45 A46 L41 V42 E43 G44 T45 A46

T45H!

T45H"

G44H"

G44H"L41H"

L41H#

V42H"

V42H!

E43H"

E43H!

E43H#

HN(CO)CAHNCA

H(CCCO)NHHBHA(CBCACO)NH

A40H"

A40H#

1H(ppm)

1H (ppm)

678910

114

116

118

120

122

124

126

128

1H (ppm)

15N(ppm)

T10

E7E7E29

G44L35 L65

T45

A66

K49

K37K23 K24E39

E58

V15V55V52

L22T19E63 Q53E36

Y60

L51K30K61

K20

V18

A21A54

V28A50

M9S34

G59C14

A46V25

G38V6

A17

A40

L4V42

K5

L41

A62V33

L2E43

G8E32

V64K3

M16D47

E56V31E57

13C(pp

Residues

m)

b c

a

50 60 70

50

100

150

200

0 10 20 30 40

d

!ave

(Hz)

G27

0

Supplementary Figure 2 | Backbone and side-chain resonance assignments of TTHA1718 in living E. coli cells. a, 2D 1H-15N HSQC spectrum of TTHA1718 in living E. coli cells. Cross peaks are labelled with their corresponding backbone assignments. b, Overlaid 1HN-13C" cross-sections of the 3D HNCA (black) and the HN(CO)CA (red) spectra


corresponding to the 15N frequencies of residues from Leu41 to Ala46. Sequential connectivities are represented by dashed red lines. c, Overlaid 1HN-1H 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 1HN and 15N nuclei between in-cell and in vitro conditions. The weighted shift difference !ave for each amino acid residue was calculated as [(!1HN)2 + (!15N)2]1/2 where !1HN and !15N are the chemical shift differences (Hz; 1 ppm corresponds to 600.13 Hz for 1H and 60.81 Hz for 15N) between the two conditions. The residues in which 1H-15N 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.


0.20.40.60.81.01.21.41.6

18

20

22

24

26

280.20.40.60.81.01.21.41.6 0.20.40.60.81.01.21.41.6

13C(ppm)

1H (ppm) 1H (ppm) 1H (ppm)

a b c

0.20.40.60.81.01.21.41.6

18

20

22

24

26

281H (ppm)

13C(ppm)

A17#

A50#

A54#A21#

A66#V6$

V15$V6$'

V$V18$'A46#

A62#

V$V$V64$'

V64$ V$ V33$V28$

L51%L35%V18$

L22%L51%'L%

L35%'

L22%'

V31$L65%L%

L%L65%'V28$'L%

V$V15$'

V$

V$A40#

V25$'

d

8.58.67.47.57.47.5

18

20

22

24

26

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

13C(ppm)

1H(ppm)

8.98.58.68.58.6 9.0

18

20

22

24

26

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

13C(ppm)

1H(ppm)

18

20

22

24

26

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

13C(ppm)

1H(ppm)

1H (ppm)

L51HN L51HN E32HN E32HN E36HN L35HN

A50C#

A50H#

V31C$

V31H$

L35C%

L35C%'

L35H%

L35H$H#

e

Supplementary Figure 3 | Assignments of side chain methyl groups of TTHA1718 in living E. coli cells. 2D 1H-13C HMQC spectra of TTHA1718 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 1H-13C correlation cross peaks. d, 2D 1H-13C HMQC spectrum of TTHA1718 in living E. coli cells. Cross peaks are labelled with their corresponding assignments. e, Overlaid 1HN-13C" or 1HN-1H 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 15N-separated NOESY-HSQC (blue) spectra used for the assignments of side-chain methyl groups of A50 C#, V31 C$ and L35 C%/C%’.


12345678

15

20

25

30

35

40

45

1H (ppm)

13C(ppm)

Supplementary Figure 4 | Background 1H-13C correlation cross peaks originating from uniform 13C-labelling. Overlay of the 1H-13C HSQC spectra of purified TTHA1718 (black) and E. coli cells expressing TTHA1718 (red).


Table 1 | NMR structure statistics for TTHA1718a

Quantity in-cell in-cell w/o ALVb in vitro

Short/medium/long-range distance restraints* 179/24/89 154/18/24 770/341/578

Restrained hydrogen bonds 23 23 —

Dihedral angle restraints 90 90 —

Maximal distance restraint violation (Å) 0.13 ± 0.01 0.11 ± 0.01 0.11 ± 0.01

Maximal dihedral angle restraint violation (º) 2.2 ± 0.5 2.9 ± 0.7 —

Deviations from idealized geometry:

Bond lengths (Å) 0.0135 ± 0.0001 0.0136 ± 0.0001 0.0133 ± 0.0001

Bond angles (º) 1.75 ± 0.04 1.79 ± 0.04 1.69 ± 0.04

AMBER energy (kcal/mol) -2496 ± 100 -2242 ± 114 -2600 ± 72

AMBER van der Waals energy (kcal/mol) -162 ± 13 -134 ± 9 -224 ± 6

Ramachandran plot statistics (%) 92/7/1/0 90/9/1/0 93/7/0/0

Backbone RMSD (Å) 0.96 ± 0.20 5.46 ± 1.02 0.34 ± 0.04

All heavy atom RMSD, Å 1.53 ± 0.21 6.18 ± 1.11 0.83 ± 0.05

Backbone RMSD to the in vitro structure (Å)c 1.16 5.55 —

All heavy atom RMSD to the in vitro structure (Å)d 1.87 6.21 —

aWhere applicable, the average value and the standard deviation over the 20 energy-refined conformers that represent the NMR structure are given. bStatistics for TTHA1718 calculated without NOE-derived distance restraints involving methyl groups obtained in methyl-selectively protonated in-cell NMR samples. cBackbone RMSD of the mean structure of the ensemble to the in vitro mean structure. dAll heavy atom RMSD of the mean structure of the ensemble to the in vitro mean structure.


6.57.07.58.08.59.09.5

114

116

118

120

122

124

126

128

6.57.07.58.08.59.09.5

E7 E29

G44

L35L65

T45

A66K49

K37K23 K24E39

E58

V15

V55

V52

L22T19E63

Q53E36

Y60L51

K30K61

K20

V18

A21A54

V28

A50

M9 S34

G59

A46

V25G38V6

A17

A40

L4V42

K5

L41

A62V33

L2E43

G8E32

V64K3

M16D47

E56E57

G27

S14

S11

E7E29

G44

L35L65

T45

A66K49

K37

K23K24E39

E58

V15

V55

V52

L22T19E63

Q53E36

Y60L51

K30K61K20

V18

A21 A54

V28 A50

M9 S34

G59

A46

V25G38V6

A17

A40

L4V42

K5

L41

A62V33

L2E43

G8E32

V64K3

M16D47 E56

E57

G27

A14

A11V31 V31

1H (ppm) 1H (ppm)

15N(ppm)

a b

50 60 70

50

100

150

200

0 10 20 30 40

!ave

(Hz)

050 60 70

50

100

150

200

0 10 20 30 40

!ave

(Hz)

0

ResiduesResidues

114

116

118

120

122

124

126

128

15N(ppm)

c d

Supplementary Figure 5 | Backbone resonance assignments of two TTHA1718 mutants, C11S/C14S and C11A/C14A, in living E. coli cells and in vitro. a, Overlay of the 2D 1H-15N HSQC spectra of TTHA1718(C11S/C14S) mutant in living E. coli cells (red) and in vitro (black). b, Overlay of the 2D 1H-15N HSQC spectra of TTHA1718(C11A/C14A) 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 Thr10, Asn12 and His13. Plots of the chemical shift differences of backbone 1HN and 15N nuclei of the C11S/C14S mutant (c) and the C11A/C14A mutant (d) between in-cell and in vitro conditions. The shift difference !ave for each amino acid residue was calculated as in supplementary Fig. 2d. The residues for which 1H-15N 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.


114

116

118

120

122

124

126

128


15N(ppm)

V15

C14

T10

M9

a b c

67891011 67891011 67891011

C11

A17

Supplementary Figure 6 | In vitro characterisation of the metal-binding activity of two TTHA1718 mutants, C11S/C14S and C11A/C14A. 2D 1H-15N HSQC spectra of wild type TTHA1718 (a), C11S/C14S (b) and C11A/C14A (c). For each panel, two spectra measured in M9 medium (red) and in M9 medium supplemented with an excess of a metal salts solution (ZnSO4, MnSO4 and CuSO4) (black) are overlaid. The final concentrations of these three metal ions were 200 &M, 50 &M and 35 &M, 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 TTHA1718, significant line broadening and/or chemical shift changes were found for residues distributed around the putative metal-binding loop (indicated in a), while no significant changes were found for the C11S/C14S or C11A/C14A mutants, suggesting that these two mutants lack metal-binding activity.


678910

114

116

118

120

122

124

126

128

678910 6789101H (ppm) 1H (ppm) 1H (ppm)

15N(ppm)

a b c

V15

C14

T10

M9

Supplementary Figure 7 | Characterisation of the metal-binding activity of two TTHA1718 mutants, C11S/C14S and C11A/C14A in E. coli cells. 2D 1H-15N HSQC spectra of wild type TTHA1718 (a), C11S/C14S (b) and C11A/C14A (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 (ZnSO4, MnSO4 and CuSO4) (black) overlaid. The final concentrations of the three metal ions were 200 &M, 50 &M and 35 &M, respectively. The metal mixture was added into the E. coli culture an hour before the cells were harvested. For wild type TTHA1718 in E. coli cells in the presence of excess Zn2+, Mn2+ and Cu2+ ions in the medium, additional line broadening and chemical shift changes similar to those seen in the in vitro experiments (supplementary Fig. 6a) were observed while no significant change was found for either mutant.


Supplementary Figure 8 | The contribution of long-range NOEs involving methyl groups to the structure calculation of TTHA1718 in living E. coli cells. a, A superposition of the 20 final structures of TTHA1718 in living E. coli cells, showing the backbone (N, C", C’) atoms. b, A superposition of the 20 final structures of TTHA1718 in living E. coli cells calculated without distance restraints derived from NOEs involving methyl groups obtained in methyl-selectively protonated in-cell NMR samples.


V33 S34 E39E32

1H (ppm)8.5

L41HN

V31H"

V42H"

H"

V31H$

H$H#

1

2

3

4

5

6

7

8

9

1H(ppm)

8.5 8.5 8.5

E32H"

H#

H$

1

2

3

4

5

6

7

8

9

1H(ppm)

8.5 8.5 8.5

V33H"

H"

A40H"

H#

V33H#

V33H$

G38H"

A40H#

8.5 8.5

1

2

3

4

5

6

7

8

9

1H(ppm)

7.0

S34HN

G38HN

H"

G38H"

V33H$

H#

H#'

H$

S34H#

K37H"

K37H#

7.0 7.0

1

2

3

4

5

6

7

8

9

1H(ppm)


3 hr 1 hr 0.5 hr 3 hr 1 hr 0.5 hr 3 hr 1 hr 0.5 hr 3 hr 1 hr 0.5 hr

678910

114

116

118

120

122

124

126

128

678910

3 hr 1 hr 0.5 hr

1H ( )1H (ppm) 1H (ppm)

15N(ppm)

a b c

d e f

g

E32

E32 E32

ppm678910

Supplementary Figure 9 | 3D 15N-separated NOESY-HSQC spectra acquired on TTHA1718 in-cell NMR samples with various protein expression levels. The concentration of TTHA1718 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 1H-15N HSQC spectra are shown for the in-cell NMR samples with three different incubation times, 3 hours (d), 1 hour (e) and 30 minutes (f) prior to cell harvest. 1D cross


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 TTHA1718 in the in-cell NMR samples collected after 1 hour or 30 minutes were estimated to be 1.2-1.6 and 0.6-0.8 mM, respectively. g, 1HN-1H cross-sections corresponding to the 15N frequencies of residues, Glu32, Val33, Ser34 and Glu39 extracted from the 3D 15N-separated NOESY-HSQC spectra of TTHA1718 in-cell NMR samples with incubation times of 3 hours, 1 hour and 30 minutes prior to cell harvest. All three 3D 15N-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. Intra-residue NOEs are indicated by blue boxes and annotated. Even for the samples with 1 hour and 30 minutes incubation time, we could identify 74% (364) and 61% (299) of all cross peaks (487) observed for the sample with 3 hours’ incubation time and used for the structure calculation.


6.57.07.58.08.59.0

112

114

116

118

120

122

124

126

128

1H (ppm)

15N(ppm)

8.58.6

54

60

56

58

62

13C(ppm)

1H (ppm)7.98.08.28.37.57.68.18.28.78.8

T44 E45 A46 E47 L48 Q49

HN(CO)CAHNCA

a b c

T5

E45A46

E47L48

Q49

8.08.1

0

1

2

3

4

5

6

7

8

9

8.48.5 8.78.8 8.48.5 7.57.6

L4 T5 E6 E7 Q8

1H (ppm)

1H(ppm)

L4

T44

E6

E7

Q8

Supplementary Figure 10 | In-cell NMR spectra of rat calmodulin in E. coli cells. a, 2D 1H-15N HSQC spectrum of rat calmodulin in E. coli JM109 (DE3) cells. The concentration of calmodulin in in-cell NMR samples was estimated to be 1.0-1.5 mM by SDS-PAGE. Note that 1H-15N HSQC spectra with equivalent quality were measured when using HMS174(DE3) as host E. coli cells, while 1H-15N correlation cross peaks were extremely broadened when using BL21(DE3) as host cells. b, Overlaid 1HN-13C" cross-sections of the 3D HNCA (black) and the HN(CO)CA (red) spectra corresponding to the 15N 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 1H-15N-HSQC are indicated in panel a. c, 1HN-1H cross-sections corresponding to the 15N frequencies of residues from Leu4 to Gln8 extracted from the 3D 15N-separated NOESY-HSQC spectrum. Sequential connectivities of HN-HN 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.


K5

K3

K61

K37

K20

K23

K24K30

K49i N-term.

C-term.

0 200 400 600 800 1000 1200 14000.0

0.2

0.4

0.6

0.8

1.0 K3K5K20/K49K24K30K37/K61

K3K5K20/K49K24K30K37/K61

678910

114

116

118

120

122

124

126

128

100 120 140 160 180 200 220 240806040200

K5 K3

K20

K49

K61

K37

K23K24

K30

*

K5K3

K20/K49K23/*K37/K61

K30 K24 K5 K3

K20K49

K61K37K23 K24

K30

678910

114

116

118

120

122

124

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128

K5 K3

K20

K49

K61

K37

K23K24

K30

0.0

0.4

0.6

0.8

1.0

0.0

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0 200 400 600 800 1000 1200 1400

100 120 140 160 180 200 220 240806040200

0.2

0.2

Time (ms) Time (ms)

Time (ms) Time (ms)

Inte

nsity

Inte

nsity

Inte

nsity

Inte

nsity

1H (ppm)1H (ppm)

15N(ppm)

15N(ppm)

K3K5K20K23K24K30K37K49K61

K3K5K20K23K24K30K37K49K61

a bc d

e f

g h

Supplementary Figure 11 | Longitudinal (T1) and transverse (T2) 15N relaxation data of TTHA1718 in living E. coli cells. 15N relaxation data in living E. coli cells and in vitro were obtained by measuring 1D 15N-edited 15N T1 or T2 relaxation experiments with various relaxation delays on samples selectively labelled with 15N-lysine. Each relaxation experiment was repeated 4-5 times for statistical analysis. The 2D 1H-15N HSQC spectrum (c) and its 1D projection (a) of lysine selectively 15N-labelled TTHA1718 in living E. coli cells are shown. Corresponding 2D and 1D spectra measured in vitro are shown in d and b, respectively. 15N T1 and T2 data for the backbone amide 15N nuclei of lysine residues of TTHA1718 in E. coli cells (e and g) and in vitro (f and h) are displayed with their single-exponential least-squares best-fit curves. Error bars, if not shown, lie within the size of symbols used to indicate the data points. T1 values were obtained by using 11 relaxation


delays of 15, 55, 105, 155, 255, 405, 505, 755, 1005, 1205 and 1505 ms. T2 values were obtained using 5 relaxation delays (14.4, 28.8, 43.2, 72.0 and 100.8 ms) for in-cell samples and 6 relaxation delays (14.4, 43.2, 72.0, 115.2, 172.8 and 230.4 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 TTHA1718 is shown in panel i.


Table 2 | 15N T1 and T2 relaxation times for backbone 15N nuclei of lysine residues of TTHA1718 in E. coli cells and in vitro, at a spectrometer frequency of 600 MHz and a temperature of 310 K.

Relaxation times (mean and s.d.) Resdue

T1 (ms), in-cell T1 (ms), in vitro T2 (ms), in-cell T2 (ms), in vitro

Lys3 835 ± 15 466 ± 3 44.3 ± 3.1 201 ± 2

Lys5 767 ± 64 431 ± 26 47.8 ± 2.8 193 ± 1

Lys20 413 ± 3 174 ± 1

Lys20/Lys49 807 ± 18 43.5 ± 1.2

Lys23 433 ± 1 184 ± 1

Lys24 763 ± 42 429 ± 3 36.8 ± 2.2 187 ± 1

Lys30 939 ± 76 525 ± 2 41.9 ± 5.2 223 ± 1

Lys37 424 ± 5 191 ± 2

Lys37/Lys61 946 ± 32 52.2 ± 2.1

Lys49 453 ± 1 190 ± 1

Lys61 536 ± 7 230 ± 2


nature template - pc word 97 · 2020-03-06 · 2d 1h-15n hsqc spectra of a ttha1718 in-cell nmr...

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