Figure 1.
Pseudo-4D TREDOR (H)(N)CNH and (H)N(H)CH pulse sequences shown in (A,B), respectively. Narrow and wide filled rectangles represent hard π/2 and π pulses, respectively, while soft selective pulses on the carbon channel are shown as parabolas. The REDOR periods are implemented with the usual two hard π pulses32,33 per rotor period applied to the carbon channel. REDOR pulses were cycled according to the xy-8 scheme.55 In the middle of each REDOR period, a REBURP59-shaped pulse is used to select the 13C chemical shifts of interest. For 15N–13Cα TREDOR, the bandwidth of this pulse is set to 40–70 ppm; for 15N–13Cx TREDOR, the bandwidth of this pulse is set to 5–53 ppm, which should refocus all side-chain 13C shifts except for that of the Cβ of serine and threonine; for H–CO TREDOR, the bandwidth of this pulse is set to 154–254 ppm. In (A), water suppression57 is split before and after acquisition of the second dimension (t2). This doubles as two z-filter elements, allowing a reduced phase cycle. Pulses were phase cycled as Φ1 = 11333311, Φ2 = 00222200, Φ3 = 01, Φ5 = 0022, Φ6 = 1133, Φ7 = 0022, Φ8 = 2200, Φ9 = 0022, Φ10 = 2, Φrec′r = 02202002, where 0 = x, 1 = y, 2 = −x, 3 = −y. Unless indicated, other pulses have phase 0. To conveniently encode a correct ppm scale during acquisition of the first indirect dimension in (A) for both 15N and 13C, the increment of the 13C shifts t1C is set to t1N × (γ15N/γ13C), where γ is the gyromagnetic ratio, so that 15N and 13C will share the same ppm scale. Hypercomplex data were acquired by shifting Φ7, Φ8, Φ10 by −90°, and Φ9 by +90° in a separate acquisition to acquire pure phases, according to the procedure of Ruben and co-workers.72