The 1D 15N-13C spectra were obtained at a spinning frequency of 13.0 kHz, on a custom-built spectrometer (courtesy of Dr. D. Ruben, Francis Bitter Magnet Laboratory/MIT, Cambridge, MA) operating at 750 MHz 1H Larmor frequency and equipped with a triple-resonance 1H/13C/15N 3.2 mm E-free probe (Bruker Biospin, Billerica MA).
The NCO SPECIFIC-CP condition was optimized to match 2.5 times the rotor frequency (ωr) on 15N (~32.5 kHz) and 3.5 × ωr on 13C (45.5 kHz), with 100 kHz 1H CW decoupling during the transfer. The 13C carrier was set to the middle of the CO region (176 ppm), the 15N carrier to 115 ppm, and the 1H carrier to 4 ppm.
The NCa SPECIFIC-CP condition was optimized to match 1.5 × ωr on 15N and 2.5 × ωr on 13C, with 100 kHz 1H CW decoupling during the transfer. The 13C carrier was set to 57 ppm, the 15N carrier to 115 ppm, and the 1H carrier to 4 ppm. The optimal NCa contact time was found to be 6 ms for both GB1 and GvpA.
Broadband DCP was optimized for overall (both NCa and NCO) transfer efficiencies. This caused suboptimal NCO and NCa transfers individually, but gave the overall highest simultaneous signal. To achieve this, the 13C carrier was set to 110 ppm, with radio frequency matching conditions of 2.5 × ωr on 15N (~32.5 kHz) and 3.5 × ωr on 13C (45.5 kHz), and 100 kHz 1H CW decoupling during the transfer. The optimal DCP contact time was found to be 7 ms for both GB1 and GvpA.
The ZF-TEDOR experiments were performed using 50 kHz for both 13C and 15N. The mixing period was optimized to 1.28 ms for one bond 15N-13C transfer. (Jaroniec et al. 2002b (link)).
For all 1D comparisons, 83 kHz TPPM 1H decoupling was used during acquisition (total phase difference,18°; TPPM pulse length 5.8 μs). Chemical shifts were referenced using the DSS scale (Morcombe and Zilm 2003 (link)), with adamantane (40.48 ppm for 13C) as a secondary standard. Relative NCO transfer efficiencies were determined by integrating the region from 170 ppm to 182 ppm (omitting the carboxyl peaks) for GB1 and GV, while relative NCa transfer efficiencies were determined by integrating the region from 50 ppm to 63 ppm for GV and 47 ppm to 63 ppm for GB1, assuring that only polarization from Ca carbons was used to evaluate transfer efficiencies.
The NCO SPECIFIC-CP condition was optimized to match 2.5 times the rotor frequency (ωr) on 15N (~32.5 kHz) and 3.5 × ωr on 13C (45.5 kHz), with 100 kHz 1H CW decoupling during the transfer. The 13C carrier was set to the middle of the CO region (176 ppm), the 15N carrier to 115 ppm, and the 1H carrier to 4 ppm.
The NCa SPECIFIC-CP condition was optimized to match 1.5 × ωr on 15N and 2.5 × ωr on 13C, with 100 kHz 1H CW decoupling during the transfer. The 13C carrier was set to 57 ppm, the 15N carrier to 115 ppm, and the 1H carrier to 4 ppm. The optimal NCa contact time was found to be 6 ms for both GB1 and GvpA.
Broadband DCP was optimized for overall (both NCa and NCO) transfer efficiencies. This caused suboptimal NCO and NCa transfers individually, but gave the overall highest simultaneous signal. To achieve this, the 13C carrier was set to 110 ppm, with radio frequency matching conditions of 2.5 × ωr on 15N (~32.5 kHz) and 3.5 × ωr on 13C (45.5 kHz), and 100 kHz 1H CW decoupling during the transfer. The optimal DCP contact time was found to be 7 ms for both GB1 and GvpA.
The ZF-TEDOR experiments were performed using 50 kHz for both 13C and 15N. The mixing period was optimized to 1.28 ms for one bond 15N-13C transfer. (Jaroniec et al. 2002b (link)).
For all 1D comparisons, 83 kHz TPPM 1H decoupling was used during acquisition (total phase difference,18°; TPPM pulse length 5.8 μs). Chemical shifts were referenced using the DSS scale (Morcombe and Zilm 2003 (link)), with adamantane (40.48 ppm for 13C) as a secondary standard. Relative NCO transfer efficiencies were determined by integrating the region from 170 ppm to 182 ppm (omitting the carboxyl peaks) for GB1 and GV, while relative NCa transfer efficiencies were determined by integrating the region from 50 ppm to 63 ppm for GV and 47 ppm to 63 ppm for GB1, assuring that only polarization from Ca carbons was used to evaluate transfer efficiencies.
