Development of Solid State NMR Methods and Technology

2022财年在以下方面取得了进展： (1)新的快速混合/冷冻技术在钙调蛋白/多肽复合体形成中的应用。在2022财年，我们完成并发表了一项关于钙负载钙调蛋白与肌球蛋白轻链激酶靶肽(M13肽)形成复合体的分子机制的研究。这项与Clore团队在LCP的合作研究包括脉冲EPR和固态核磁共振测量的新组合，并利用了我们实验室在过去几年开发的快速混合和快速冷冻捕获技术。脉冲EPR结合钙调蛋白的双自旋标记，提供了关于钙调蛋白结构的时间依赖性的信息，而固体核磁共振提供了关于M13构象的时间依赖性和M13-钙调蛋白接触的时间依赖性的信息。结合的数据很好地符合动力学模型，根据该模型，两条不同的途径导致最终的钙调蛋白/钙/M13复合体。在一个途径中，M13首先与钙调蛋白的C-末端结构域结合，然后在大约0.5ms内进入最终的复合体。在另一种途径中，M13首先与钙调蛋白的N-末端结构域结合，然后进入中间状态，在该状态下钙调蛋白发生了构象变化，但M13尚未完全有序。这种中间状态进行到最终复合体的速度相对较慢，大约为6毫秒。这项研究发表在《美国国家科学院院刊》上。这是第一个用互补的时间分辨固体核磁共振和EPR测量表征生物分子络合物形成机理的例子，并根据一个特定的动力学模型进行了分析，在这个模型中，各种状态都有特定的结构特征。 (2)逆温跳跃核磁共振：作为时间分辨固体核磁共振快速混合的替代方法，我们开发了一种简单的方法，可以在0.6毫秒内将蛋白质溶液的温度从90℃改变到30℃。这种方法包括使溶液流经铜毛细管，铜毛细管焊接到热铜块和冷铜块上。在2022财年，我们完成了对蜂毒肽蜂毒素的一系列实验，蜂毒素在90℃下从主要单体状态转变为30℃下的四聚状态。时间分辨固体核磁共振数据在1-30毫秒范围内的演化时间表明，在快速反向温度跃升后，可以监测到四聚过程。对含时2D固体核磁共振谱的分析表明，蜂毒素单体形成螺旋的时间尺度和分子间接触形成的时间尺度几乎相同，符合单向二聚模型。因此，螺旋的形成和二聚体的形成是一个协调的过程，四聚体在二聚后迅速形成。这些结果，包括对新型逆温跃变装置的完整描述，发表在《美国医学会杂志》上。 (3)微米级磁共振成像：利用我们实验室近年来发展起来的低温磁共振成像(MRI)和动态核极化(DNP)技术，我们已经在极低的温度(5开尔文)下获得了第一个DNP增强的MRI图像，由此产生的核磁共振信号的增强可以显著提高图像分辨率。具体地说，我们已经获得了测试样品的图像，该样品由9微米直径的玻璃珠组成，在40微米内径的毛细管中浸泡在甘油/水中。这些图像的名义分辨率为1.7微米(在所有三个空间维度上)。在2022财年，我们完成了对实验图像的定量分析，结果表明，真实分辨率与标称分辨率大致相同(绝对优于2微米的各向同性分辨率)。这是MRI分辨率的新世界纪录。一篇描述这些结果的论文，包括对新型低温核磁共振设备的完整描述，发表在《美国国家科学院院刊》上。 (4)用于动态核极化的新的三核素化合物：我们使用动态核极化(DNP)在上述时间分辨固体核磁共振和低温核磁共振实验中产生大信号增强。DNP依赖于向核磁共振和核磁共振样品中添加顺磁性化合物。前些年，我们发现在很低的温度下(低于30K)，含有三个氮氧自由基基团的化合物对DNP特别有利。在2022财年，我们合成并测试了这些三氮氧化合物的两个新变种，设计为在我们实验室的各种实验所需的广泛pH值范围内具有高溶解度。合成方法及其在25K下DNP增强固体核磁共振中的有效性的演示发表在《磁共振杂志》上。

Progress in FY2022 was in the following areas: (1) APPLICATION OF NEW RAPID MIXING/FREEZING TECHNOLOGY TO CALMODULIN/PEPTIDE COMPLEX FORMATION. In FY2022, we completed and published a study of the molecular mechanism by which calcium-loaded calmodulin forms a complex with a target peptide from myosin light chain kinase (M13 peptide). This collaborative study with the Clore group in LCP included a novel combination of pulsed EPR and solid state NMR measurements and took advantage of rapid mixing and rapid freeze-trapping technology developed in our laboratory over the past several years. Pulsed EPR, combined with double spin labeling of calmodulin, provided information about the time dependence of the calmodulin structure, while solid state NMR provided information about the time dependence of the M13 conformation and the time dependence of M13-calmodulin contacts. The combined data were well fit with a kinetic model, according to which two separate pathways lead to the final calmodulin/Ca2+/M13 complex. In one pathway, M13 binds first to the C-terminal domain of calmodulin, then proceeds to the final complex within about 0.5 ms. In the other pathway, M13 binds first to the N-terminal domain of calmodulin, then proceeds to an intermediate state in which calmodulin has undergone a conformational change but M13 is not yet fully ordered. This intermediate state proceeds to the final complex relatively slowly, in about 6 ms. This work was published in PNAS. It is the first example in which a biomolecular complex formation mechanism has been characterized by complementary time-resolved solid state NMR and EPR measurements and analyzed in terms of a specific kinetic model in which the various states have specific structural characteristics. (2) INVERSE TEMPERATURE-JUMP NMR: As an alternative approach to rapid mixing for time-resolved solid state NMR, we have developed a simple method for changing the temperature of a protein solution from 90 C to 30 C in about 0.6 milliseconds. This method involves flowing the solution through copper capillary tubes that are soldered to hot and cold copper blocks. In FY2022, we completed a series of experiments on the bee venom peptide melittin, which converts from a largely monomeric state at 90 C to a tetrameric state at 30 C. Time-resolved solid state NMR data for evolution times in the 1-30 millisecond range demonstrated that the tetramerization process can be monitored after a rapid inverse temperature jump. Analyses of time-dependent 2D solid state NMR spectra showed that time scales for helix formation by melittin monomers and for the development of intermolecular contacts are nearly identical and can be fitted to a unidirectional dimerization model. Thus, helix formation and dimer formation occur as a concerted process, and tetramers form rapidly after dimerization. These results, including a full description of the novel inverse temperature-jump apparatus, were published in JACS. (3) MICRON-SCALE MRI: Using low-temperature magnetic resonance imaging (MRI) and dynamic nuclear polarization (DNP) technology developed in our lab in recent years, we have obtained the first DNP-enhanced MRI images at very low temperatures (5 Kelvin), where the resulting enhancements of NMR signals can permit significant improvements in image resolution. Specifically, we have obtained images of a test sample, consisting of 9 micron-diameter glass beads immersed in glycerol/water within a 40 micron-inner-diameter capillary. The nominal resolution of these images is 1.7 microns (in all three spatial dimensions). In FY2022, we completed a quantitative analysis of the experimental images, which indicates that the true resolution is approximately equal to the nominal resolution (definitely better than 2 micron isotropic resolution). This is a new world's record for resolution in MRI. A paper describing these results, including a full description of the novel low-temperature MRI apparatus, was published in PNAS. (4) NEW TRIRADICAL COMPOUNDS FOR DYNAMIC NUCLEAR POLARIZATION: We use dynamic nuclear polarization (DNP) to produce large signal enhancements in the time-resolved solid state NMR and low-temperature MRI experiments described above. DNP depends on addition of paramagnetic compounds to the NMR and MRI samples. In previous years, we showed that compounds containing three nitroxide free radical moieties are particularly favorable for DNP at very low temperatures (below 30 K). In FY2022, we synthesized and tested two new variants of these tri-nitroxide compounds, designed to have high solubilities over a wide range of pH values as required for various experiments in our lab. The synthesis methods and demonstrations of their effectiveness in DNP-enhanced solid state NMR at 25 K were published in the Journal of Magnetic Resonance.