An extended combinatorial 15N, 13C, and 13C’ labeling approach to protein backbone resonance assignment

F. Löhr1, F. Tumulka2, C. Bock2, R. Abele2, V. Dötsch1

1Institute of Biophysical Chemistry & Center for Biomolecular Magnetic Resonance, Goethe

University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany

2Institute of Biochemistry, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany

Supplementary Material

Fig. S1 Full set of 2D 1H-15N correlation spectra recorded on the three combinatorial 15N, 13C, 13C’ labeled samples of TMD0. HN(CO) and HN(COCA) spectra were recorded at 600 MHz, all remaining experiments were recorded at 800 MHz as described in the main text. For clarity, in the HSQC spectra (top row) assignments are indicated only for those residues not observed in any of

13 13 15 the triple-resonance spectra. Empty boxes indicate the positions of cross peaks from 2- Ci-1- C/ Ni labeled amino acid pairs that were not detected in CO-filtered HN(CA) spectra. A circle is drawn at the position of the backbone resonance of W115 (sample 2) which was expected to be observed in the control spectrum of the CO-filtered HN(CA) but had insufficient signal-to-noise. Cross peaks appearing at 13C natural abundance are labeled with residue identifiers followed by a dagger. An unassigned resonance of a leucine residue in sample 3, possibly arising from a minor conformer, is marked with an asterisk. The label Wsc in the HSQC of sample 2 denotes a tryptophan side-chain (NH) correlation. Bruker pulse sequence code (tested for TopSpin 2.1 and TopSpin 3.1 on Bruker Avance and Avance II spectrometers, respectively) and acquisition parameters (800 MHz) for the carbonyl-filtered BEST-[15N,1H]-TROSY-HN(CA) experiment

;b_hnca_cofilt_tr2d.fl

;carbonyl-filtered [15N,1H]-BEST-TROSY-HN(CA) for distinction of ;13Ca(i-1)-12C'(i-1)--15N(i)-12Ca(i)-12C'(i) and 12Ca(i-1)-12C'(i-1)-15N(i)-13Ca(i)-13C'(i) moities ;interleaved recording of HN(CA) w and w/o 1JCaC' evolution during Ca spin-echo

#include #include #include

"p22=p21*2"

"p11=3.873/(4*(bf2*cnst22/1000000-o2))" "p12=1.732/(2*(bf2*cnst22/1000000-o2))"

"d5=2.7m-d4+p42/2" ; =2.7m-p41*cnst41-p17-200u-DELTA1

"d25=3u" "d15=d20-p12-d25" "d16=d20-p18+p21*1.273"

"in0=inf2/4" "in20=(d20-10u)/(td2/2-1)" "if ( in20 > in0) { in25=0; } else { in25=in0*(1-in20/in0)*2; }" "if ( in20 > in0) { in15=in0; } else { in15=in20; }" "if ( in20 > in0) { in16=in0; } else { in16=in0*(2-in20/in0); }" "if ( in20 > in0) { in20=in0; }"

"DELTA1=d4-p17-200u-p41*cnst41-p42/2" "DELTA2=d3/2-p17-200u" "DELTA3=d2/2-p17-200u" "DELTA5=p19+204u" "DELTA6=d8-p17-200u-p42/2-p43*0.67" "DELTA7=d8-p16-200u-p42/2"

"spoff25=bf1*(cnst19/1000000)-o1" "spoff26=bf1*(cnst19/1000000)-o1" "spoff28=bf1*(cnst19/1000000)-o1" "spoff29=bf1*(cnst19/1000000)-o1"

"spoff1=bf2*cnst22/1000000-o2" "spoff3=0" "spoff4=0" "spoff5=0" "l0=0" aqseq 312

1 d11 ze d11 pl0:f1 pl0:f2 pl3:f3 2 d11 3 d12 (p21 ph1 3u p22*1.167 ph9 3u p21 ph1):f3 d1 50u UNBLKGRAD

(p41:sp25 ph1):f1 p17:gp1 200u DELTA1 if "d4 > 2.7m" { (p42:sp26 ph1):f1 (d5 p21 ph1 3u p22*1.167 ph9 3u p21 ph1):f3 } else { (center (p42:sp26 ph1):f1 (p21 ph1 3u p22*1.167 ph9 3u p21 ph1):f3 ) } p17:gp1 200u DELTA1 (p41:sp25 ph2):f1

(p21 ph3):f3 p17:gp2 200u DELTA2 (center (p12:sp3 ph1):f2 (p22 ph1):f3 ) p17:gp2 200u DELTA2 (p21 ph1):f3 if "l0 %2 == 0" { (p11:sp4 ph4):f2 3u (p14:sp1 ph1):f2 p17:gp3 200u DELTA3 (p15:sp5 ph1):f2 p17:gp3 200u DELTA3 (p14:sp1 ph1):f2 3u (p11:sp4 ph5):f2 } if "l0 %2 == 1" { (p11:sp4 ph4):f2 3u (p14:sp1 ph1):f2 p17:gp3 200u DELTA3 (p15:sp5 ph1):f2 3u (p14:sp1 ph1):f2 p17:gp3 200u DELTA3 (p11:sp4 ph5):f2 }

(p21 ph6):f3 d20 (p14:sp1 ph1):f2 d20 (p22 ph1):f3 d25 (p12:sp3 ph1):f2 d15 (p14:sp1 ph1):f2 d16 p18:gp4*EA DELTA5

(p43:sp28 ph8):f1 p17:gp5 200u DELTA6 (center (p42:sp26 ph1):f1 (p12:sp3 ph1):f2 (p22 ph1):f3 ) p17:gp5 200u DELTA6 (p43:sp29 ph1):f1

DELTA5 (p21 ph9):f3 p16:gp6 200u DELTA7 (center (p42:sp26 ph1):f1 (p22 ph1):f3 ) p16:gp6 200u DELTA7 (p21 ph12):f3

p19:gp7 200u 4u BLKGRAD

go=2 ph31 d11 mc #0 to 2 F1QF(iu0) F2EA(igrad EA & ip8*2 & ip12*2, dd20 & id25 & id15 & id16 & ip6*2 & ip31*2) exit ph1 = 0 ph2 = 1 ph3 = 1 1 3 3 ph4 = 0 2 ph5 = 0 0 0 0 2 2 2 2 ph6 = 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 ph8 = 1 ph9 = 1 ph12= 0 ph31= 0 2 2 0 2 0 0 2 2 0 0 2 0 2 2 0

;cnst19: HN chemical shift (offset, in ppm)

;cnst21: Calpha chemical shift (offset, in ppm) ;cnst22: CO chemical shift (offset, in ppm) ;o2p: Ca chemical shift

;d2: 1/(2*1JC'Ca) ;d3: <= 1/(2*1JNCa) ;d20: <= 1/(8^1JNCa)-1/(8*1JNH)

;gradients: ;p16: 1m ;p17: 300u (200u @ 600MHz) ;p18: 1.4m ;p19: 175u

;for z-only gradients: ;gpz1: -10% ;gpz2: -15% ;gpz3: -12% ;gpz4: 78.9% ;gpz5: 8% ;gpz6: 25% ;gpz7: 64% Current Data Parameters PL1W 9.29792213 W NAME tmd0combi_800 SFO1 800.0337730 MHz EXPNO 155 SP25 30.20 dB PROCNO 1 SP26 17.00 dB SP28 20.10 dB F2 - Acquisition Parameters SP29 20.10 dB Date_ 20140905 SPNAM[25 Pc9_4_90.1000 Time 14.49 SPNAM[26 Reburp.1000 INSTRUM spect SPNAM[28 Eburp2tr.1000 PROBHD 5 mm TXI 1H/2H SPNAM[29 Eburp2.1000 PULPROG b_hnca_cofilt_tr2d.fl SPOAL25 0.500 TD 768 SPOAL26 0.500 SOLVENT H2O+D2O SPOAL28 0.500 NS 256 SPOAL29 0.500 DS 128 spoffs25 3187.26 Hz SWH 8802.817 Hz spoffs26 3187.26 Hz FIDRES 11.462001 Hz spoffs28 3187.26 Hz AQ 0.0436224 sec spoffs29 3187.26 Hz RG 1625.5 DW 56.800 usec ======CHANNEL f2 ======DE 6.50 usec NUC2 13C TE 323.0 K P11 40.11 usec CNST19 8.6999998 P12 35.87 usec CNST22 174.0000000 P14 100.00 usec CNST41 0.5290000 P15 600.00 usec D1 0.30000001 sec PL2 120.00 dB D2 0.00940000 sec PL2W 0 W D3 0.02400000 sec SFO2 201.1785671 MHz D4 0.00245000 sec SP1 3.40 dB D5 0.00100000 sec SP3 -0.60 dB D8 0.00250000 sec SP4 6.30 dB D11 0.03000000 sec SP5 11.50 dB D12 0.00002000 sec SPNAM[1] sinc1.200 D15 0.00596113 sec SPNAM[3] squa100.10 D16 0.00464647 sec SPNAM[4] squa100.10 D20 0.00600000 sec SPNAM[5] Rsnob.1000 D25 0.00000300 sec SPOAL1 0.500 IN0 0.00006175 sec SPOAL3 0.500 IN15 0.00003770 sec SPOAL4 0.500 IN16 0.00008580 sec SPOAL5 0.500 IN20 0.00003770 sec spoffs1 24140.12 Hz IN25 0.00004810 sec spoffs3 0 Hz L0 0 spoffs4 0 Hz spoffs5 0 Hz ======CHANNEL f1 ======NUC1 1H ======CHANNEL f3 ======P41 2200.00 usec NUC3 15N P42 1500.00 usec P21 36.50 usec P43 1400.00 usec P22 73.00 usec PL0 120.00 dB PL3 -3.50 dB PL1 1.00 dB PL3W 192.32073975 W PL0W 0 W SFO3 81.0759829 MHz SW 49.936 ppm ======GRADIENT CHANNEL ===== FnMODE Echo-Antiecho GPNAM[1] SINE.32 GPNAM[2] SINE.32 F3 - Acquisition parameters GPNAM[3] SINE.32 SW 49.936 ppm GPNAM[4] SINE.32 SFO1 81.07598 MHz GPNAM[5] SINE.32 HZpPT 0.494212 Hz GPNAM[6] SINE.32 TD 2 GPNAM[7] SINE.32 GPZ1 -10.00 % F2 - Processing parameters GPZ2 -15.00 % SI 1024 GPZ3 -12.00 % SF 800.0301111 MHz GPZ4 78.90 % WDW QSINE GPZ5 8.00 % SSB 2 GPZ6 25.00 % LB 0 Hz GPZ7 64.00 % GB 0 P16 1000.00 usec PC 1.00 P17 300.00 usec P18 1400.00 usec F1 - Processing parameters P19 175.00 usec SI 512 MC2 echo-antiecho SF 81.0663973 MHz WDW QSINE F1 - Acquisition parameters SSB 2 TD 256 LB 0 Hz SFO1 81.07598 MHz GB 0 FIDRES 15.814777 Hz