A novel deuterium-excited and proton-detected quadruple-resonance three-dimensional (3D) ²H_αc_αNH MAS nuclear magnetic resonance (NMR) method is presented to obtain site-specific ²H_α deuterium quadrupolar couplings from protein backbone, as an extension to the 2D version of the experiment reported earlier. Proton-detection results in high sensitivity compared to the heteronuclei detection methods. Utilizing four independent radiofrequency (RF) channels (quadruple-resonance), we managed to excite the ²H_α, then transfer deuterium polarization to its attached C_α, followed by polarization transfers to the neighboring backbone nitrogen and then to the amide proton for detection. This experiment results in an easy to interpret HSQC-like 2D ¹H–¹⁵N fingerprint NMR spectrum, which contains site-specific deuterium quadrupolar patterns in the indirect third dimension. Provided that four-channel NMR probe technology is available, the setup of the ²H_αc_αNH experiment is relatively straightforward, by using low power deuterium excitation and polarization transfer schemes we have been developing. To our knowledge, this is the first demonstration of a quadruple-resonance MAS NMR experiment to link ²H_α quadrupolar couplings to proton-detection, extending our previous triple-resonance demonstrations. Distortion-free excitation and polarization transfer of ∼160–170 kHz ²H_α quadrupolar coupling were presented by using a deuterium RF strength of ∼20 kHz. From these ²H_α patterns, an average backbone order parameter of S = 0.92 was determined on a deuterated SH3 sample, with an average η = 0.22. These indicate that SH3 backbone represents sizable dynamics in the microsecond timescale where the ²H_α lineshape is sensitive. Moreover, site-specific ²H_α T₁ relaxation times were obtained for a proof of concept. This 3D ²H_αc_αNH NMR experiment has the potential to determine structure and dynamics of perdeuterated proteins by utilizing deuterium as a novel reporter.

提出了一种新的氘激发和质子检测四重共振三维 (3D) ² H _α c _α NH MAS 核磁共振 (NMR) 方法，以从蛋白质主链获得位点特异性² H _α氘四极偶联，如对先前报告的实验的 2D 版本的扩展。与异核检测方法相比，质子检测具有更高的灵敏度。利用四个独立的射频 (RF) 通道（四重共振），我们设法激发了² H _α，然后将氘极化转移到其附着的 C _α，然后极化转移到相邻的主链氮，然后转移到酰胺质子进行检测。这个实验产生了一个易于解释的类似 HSQC 的 2D ¹ H– ¹⁵ N 指纹 NMR 谱，其中包含间接三维中的位点特异性氘四极模式。如果可以使用四通道 NMR 探针技术，则² H _α c _α NH 实验的设置相对简单，通过使用我们一直在开发的低功率氘激发和极化转移方案。据我们所知，这是连接² H _{α的四重共振 MAS NMR 实验的首次演示}四极耦合到质子检测，扩展了我们之前的三重共振演示。^{~160–170 kHz 2} H _α四极耦合的无失真激发和极化转移是通过使用~20 kHz 的氘 RF 强度呈现的。根据这些² H _α模式，在氘代 SH3 样品上确定了 S = 0.92 的平均主链有序参数，平均 η = 0.22。这些表明 SH3 骨干在微秒时间尺度内表现出相当大的动态，其中² H _α线形是敏感的。此外，特定地点的² H _α T ₁获得松弛时间以证明概念。此 3D ² H _α c _α NH NMR 实验有可能通过利用氘作为新型报告基因来确定全氘化蛋白质的结构和动力学。