J. Am. Chem. Soc.,
An Exchange-Free Measure of 15N Transverse Relaxation: An NMR Spectroscopy Application to the Study of a Folding Intermediate with Pervasive Chemical Exchange
D. Flemming Hansen, Daiwen Yang, Haniqiao Feng, Zheng Zhou, Silke Wiesner, Yawen Bai, and Lewis E. Kay

Abstract:
A series of experiments are presented that provide an exchange-free measure of dipole-dipole 15N transverse relaxation, Rdd, that can then be substituted for 15N R1 or R2 rates in the study of internal protein dynamics. The method is predicated on the measurement of a series of relaxation rates involving 1H-15N longitudinal order, anti-phase 1H and 15N single-quantum coherences, and 1H-15N multiple quantum coherences; the relaxation rates of all coherences are measured under conditions of spin-locking. Results from detailed simulations and experiments on a number of protein systems establish that Rdd values are independent of exchange and systematic errors from dipolar interactions with proximal protons are calculated to be less than 1-2%, on average, for applications to perdeuterated proteins. Simulations further indicate that the methodology is rather insensitive to the exact level of deuteration so long as proteins are reasonably highly deuterated (>50%). The utility of the methodology is demonstrated with applications involving protein L, ubiquitin, and a stabilized folding intermediate of apocytochrome b562 that shows large contributions to 15N R1 relaxation from chemical exchange.

J. Am. Chem. Soc.,
Proton-Detected Solid-State NMR Spectroscopy of Fully Protonated Proteins at 40 kHz Magic-Angle Spinning

Donghua H. Zhou, Gautam Shah, Mircea Cormos, Charles Mullen, Dennis Sandoz, and Chad M. Rienstra*

Abstract:
Remarkable progress in solid-state NMR has enabled complete structure determination of uniformly labeled proteins in the size range of 5-10 kDa. Expanding these applications to larger or mass-limited systems requires further improvements in spectral sensitivity, for which inverse detection of 13C and 15N signals with 1H is one promising approach. Proton detection has previously been demonstrated to offer sensitivity benefits in the limit of sparse protonation or with ~30 kHz magic-angle spinning (MAS). Here we focus on experimental schemes for proteins with ~100% protonation. Full protonation simplifies sample preparation and permits more complete chemical shift information to be obtained from a single sample. We demonstrate experimental schemes using the fully protonated, uniformly 13C,15N-labeled protein GB1 at 40 kHz MAS rate with 1.6-mm rotors. At 500 MHz proton frequency, 1-ppm proton line widths were observed (500 ± 150 Hz), and the sensitivity was enhanced by 3 and 4 times, respectively, versus direct 13C and 15N detection. The enhanced sensitivity enabled a family of 3D experiments for spectral assignment to be performed in a time-efficient manner with less than a micromole of protein. CANH, CONH, and NCAH 3D spectra provided sufficient resolution and sensitivity to make full backbone and partial side-chain proton assignments. At 750 MHz proton frequency and 40 kHz MAS rate, proton line widths improve further in an absolute sense (360 ± 115 Hz). Sensitivity and resolution increase in a better than linear manner with increasing magnetic field, resulting in 14 times greater sensitivity for 1H detection relative to that of 15N detection.