Department of Structural Biology, School of Medicine, University of Pittsburgh, Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, 15261, United States.
Solid State Nucl Magn Reson. 2023 Jun;125:101861. doi: 10.1016/j.ssnmr.2023.101861. Epub 2023 Mar 21.
A novel deuterium-excited and proton-detected quadruple-resonance three-dimensional (3D) HcNH MAS nuclear magnetic resonance (NMR) method is presented to obtain site-specific H 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 H, then transfer deuterium polarization to its attached C, 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 H-N 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 HcNH 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 H quadrupolar couplings to proton-detection, extending our previous triple-resonance demonstrations. Distortion-free excitation and polarization transfer of ∼160-170 kHz H quadrupolar coupling were presented by using a deuterium RF strength of ∼20 kHz. From these H 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 H lineshape is sensitive. Moreover, site-specific H T relaxation times were obtained for a proof of concept. This 3D HcNH NMR experiment has the potential to determine structure and dynamics of perdeuterated proteins by utilizing deuterium as a novel reporter.
一种新型的氘激发和质子探测四重共振三维（3D）HcNH 魔角旋转核磁共振（NMR）方法被提出，用于从蛋白质骨架中获得特定位置的 H 氘核四极偶合，这是对早期报道的 2D 版本实验的扩展。与异核探测方法相比，质子探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），我们成功地激发了 H，然后将氘极化转移到其连接的 C，接着进行极化转移到相邻的骨架氮，然后再转移到酰胺质子进行检测。该实验得到了一个易于解释的 HSQC 样的 2D H-N 指纹 NMR 谱，其中在间接的第三维中包含特定位置的氘核四极偶合模式。只要有四通道 NMR 探头技术，HcNH 实验的设置相对简单，我们一直在开发低功率氘激发和极化转移方案。据我们所知，这是首次展示将 H 核四极偶合与质子探测联系起来的四重共振 MAS NMR 实验，扩展了我们之前的三重共振演示。通过使用约 20 kHz 的氘 RF 强度，展示了约 160-170 kHz H 核四极偶合的无失真激发和极化转移。从这些 H 模式中，在氘代 SH3 样品上确定了平均骨架有序参数 S = 0.92，平均 η = 0.22。这表明 SH3 骨架在微秒时间尺度上具有相当大的动力学，其中 H 谱线形状是敏感的。此外，还获得了用于概念验证的特定位置 H T 弛豫时间。这种 3D HcNH NMR 实验具有通过利用氘作为新型示踪剂来确定全氘代蛋白质结构和动力学的潜力。
