We expressed the proteins Twitch-2B and Twitch-6 in Toronto minimal medium prepared with 100% D2O and perdeuterated d-glucose and supplemented with the amino acid precursors α-ketobutyric acid (methyl-13C, 3,3-D2) and α-ketoisovaleric acid (3-methyl-13C, 3,4,4,4-D4), thus selectively labeling with 13C and 1H atoms only of the methyl groups of residues valine, leucine, and isoleucine while keeping the rest of the C atoms as 12C and protons as 2H (19).

First, the isotropic sample spectra were acquired in buffer A [20 mM Mops (pH 7.0), 100 mM NaCl, and 5 mM CaCl2 in 100% D2O]. Next, the proteins were dialyzed against buffer B [20 mM Mops (pH 7.0), 100 mM NaCl, and 10 mM EDTA] followed by dialysis against buffer C [20 mM Mops (pH 7.0) and 100 mM NaCl] and finally exchanged to buffer C prepared in 100% D2O containing three equivalents of dysprosium before NMR measurements. The protein concentration in the samples was approximately 0.5 mM.

The samples were tested with methyl-TROSY experiments (19, 28) (fig. S7) at 900 MHz and 1.1 GHz, and J couplings and J + RDC were determined using a J-modulated methyl-TROSY (29) experiment depicted in fig. S6 as matrices of 2048 × 128 complex data points with 96 transients per (t1) increment. The total J-modulation delays used were as follows: 4, 6, 8, 10, 12, 14, 16, 18, and 20 ms (fig. S8). NMR experiments were acquired using a 5-mm TCI (triple resonance cryoprobe with Inverse detection) on a 900-MHz spectrometer and a 3-mm TCI cryoprobe on a 1.1-GHz spectrometer, both equipped with NEO consoles (Bruker). The intensities (maximum amplitude) of the signals were extracted using CARA (computer aided resonance assignment) (30) on NMRPipe-processed experiments (31) and analyzed with Python scripts (fig. S8) following Pederson et al. (29).

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