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.

提出了一种新型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，从蛋白质骨架中获得位点特异性² H _α氘四极耦合，如下所示先前报告的实验的二维版本的扩展。与异核检测方法相比，质子检测具有高灵敏度。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 ，然后极化转移到邻近的主链氮，然后转移到酰胺质子进行检测。该实验产生了易于解释的类似 HSQC 的 2D ¹ H- ¹⁵ N 指纹 NMR 谱，其中包含间接三维空间中特定位点的氘四极模式。如果四通道 NMR 探针技术可用，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， ² H _α c _α NH 实验的设置相对简单。据我们所知，这是将² H _α四极耦合与质子检测连接起来的四重共振 MAS NMR 实验的首次演示，扩展了我们之前的三重共振演示。通过使用 ~20 kHz 的氘射频强度，实现了 ~160–170 kHz ² H _α四极耦合的无失真激发和偏振传输。 根据这些² H _α模式，在氘化 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 主链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，还获得了位点特异性² H _α T ₁弛豫时间以验证概念。该 3D ² H _α c _α NH NMR 实验具有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的潜力。