Supporting Information
Figure S1. Pulse sequence 2D V/L-CT-MQ-HmCm serves the same purpose as that of 2D V/L-CT-HmCm (Figure 1A). It separates methyl groups of Val and Leu in methyl protonated [L(13CH3, 13CH3), V(13CH3, 13CH3)]-U-[15N, 13C, 2H]-protein samples, and allows for detection of a subset of methyl groups from each residue based on the Ca chemical shift of Val or Cb chemical shift of Leu. This pulse sequence differs from that shown in Figure 1A in that the coherence of the 1H-13C of methyl groups is in the multiple quantum (MQ) state instead of single quantum state during the C-C COSY transfer steps (2tb and 2Tb periods), as used in the 4D ALA-HMCBCACO experiment (Sheppard et al. 2009). In this way, the methyl-TROSY principle can be fully exploited for the isolated protonated groups (Tugarinov and Kay 2004). The parameters of this experiment are similar to those described in Figure 1A., with the following differences. The phase cycling are f1 = x, -x; f2 = 2(y), 2(-y); f3 = f4 = 2(x), 2(-x), 2(y), 2(-y); f5 = 4(y), 4(-y); f6 = x, -x; f7 = 2(x), 2(-x); f8 = 8(x), 8(-x); rec = 8(x), 8(-x). Quadrature detection in t1 is achieved by STATES-TPPI on f8 (Marion et al. 1989). The pulsed field gradients g1–g4 are applied along the z-axis with strength (G/cm)/duration (ms) of g1 = 11/1.0, g2 = 5.5/1.0, g3 = 22/1.0, and g4 = 39.5/0.5. Similar to the parameters described in Figure 1A, the receiver phase cycling is 4(x, -x), 4(-x, x), and the I-BURP pulse (*) is moved leftward 1.65 ms for detection of the Leu methyl group.
Figure S2. The simulated I-BURP excitation profiles using the Bruker Topspin software. (A) The red curve indicates the simulated excitation profile of a 12 ms I-BURP. The complete inversion range is 334 Hz, and the 70% inversion range is 404 Hz. The black curve indicates the simulated excitation profile of a 2 ms I-BURP. The inversion range covers 13 ppm for 13C on a 600 MHz NMR instrument. (B) The overlap of two simulated 12 ms I-BURP excitation profiles with irradiation frequency ± 60 Hz from 0 Hz. The overlapped full inversion region of the two I-BURP pulses is 204 Hz, which is about 1.4 ppm for 13C on a 600 MHz NMR instrument.
Reference
Marion D, Ikura M, Tschudin R, Bax A. 1989. Rapid Recording of 2d Nmr-Spectra without Phase Cycling - Application to the Study of Hydrogen-Exchange in Proteins. J Magn Res 85: 393-399.
Sheppard D, Guo CY, Tugarinov V. 2009. 4D (1)H-(13)C NMR Spectroscopy for Assignments of Alanine Methyls in Large and Complex Protein Structures. J Am Chem Soc 131: 1364-1365.
Tugarinov V, Kay LE. 2004. An isotope labeling strategy for methyl TROSY spectroscopy. J Biomol NMR 28: 165-172.
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