Proton-Detected Solid-State NMR Spectroscopy of Fully Protonated Proteins at 40 kHz Magic-Angle Spinning

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• 作者：Donghua H. Zhou ; Gautam Shah ; Mircea Cormos ; Charles Mullen ; Dennis Sandoz ; Chad M. Rienstra
• 刊名：Journal of the American Chemical Society
• 出版年：2007
• 出版时间：September 26, 2007
• 年：2007
• 卷：129
• 期：38
• 页码：11791 - 11801
• 全文大小：411K
• 年卷期：v.129,no.38(September 26, 2007)
• ISSN：1520-5126

文摘

Remarkable progress in solid-state NMR has enabled complete structure determination ofuniformly 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 ¹³Cand ¹⁵N signals with ¹H is one promising approach. Proton detection has previously been demonstrated tooffer 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 simplifiessample preparation and permits more complete chemical shift information to be obtained from a singlesample. We demonstrate experimental schemes using the fully protonated, uniformly ¹³C,¹⁵N-labeled proteinGB1 at 40 kHz MAS rate with 1.6-mm rotors. At 500 MHz proton frequency, 1-ppm proton line widths wereobserved (500 ± 150 Hz), and the sensitivity was enhanced by 3 and 4 times, respectively, versus direct¹³C and ¹⁵N detection. The enhanced sensitivity enabled a family of 3D experiments for spectral assignmentto be performed in a time-efficient manner with less than a micromole of protein. CANH, CONH, and NCAH3D spectra provided sufficient resolution and sensitivity to make full backbone and partial side-chain protonassignments. At 750 MHz proton frequency and 40 kHz MAS rate, proton line widths improve further in anabsolute sense (360 ± 115 Hz). Sensitivity and resolution increase in a better than linear manner withincreasing magnetic field, resulting in 14 times greater sensitivity for ¹H detection relative to that of ¹⁵Ndetection.