Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins
Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins
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DOI：
10.1021/jacs.3c09184
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发表时间：
2023-11-14
影响因子：
15
通讯作者：
Rajca，Andrzej
中科院分区：
文献类型：
--
作者：
Yang，Zhimin;Stein，Richard A.;Rajca，Andrzej
本文报道了氯乙酰胺2，6-二氮杂金刚烷氮氧自由基（ClA-DZD）与葫芦[7]脲（CB-7）在水中的缔合常数Ka= 1.9 × 106 M ~（-1），比以往研究的有机自由基高至少一个数量级。自由基被CB-7高度固定，如旋转相关时间τrot相对于缓冲溶液中的旋转相关时间τrot增加36倍所示。ClA-DZD@CB-7的X射线结构显示了未扭曲的CB-7主体内的包封的DZD客体，其中侧基突出到外部。将CB-7加入到双自旋标记的T4溶菌酶（T4 L）中，我们观察到τ旋转和电子自旋相干时间Tm沿着增加，自旋间距分布变窄。在83 K的DEER测量的灵敏度增加了4-9倍，相比，常见的自旋标签，如MTSL，这是不受CB-7。3 nm的自旋间距离可以在水/甘油中可靠地测量到接近基质的玻璃化转变/熔融温度（200 K）的温度，从而使我们更接近于在接近生理温度下实现超分子聚合的长距离DEER测量的目标。DZD-T4 L 65在1.12 μ m分辨率下的X射线结构允许DZD标记的明确建模（0.88占有率），表明蛋白质的未受干扰的结构和构象。
We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water,Ka= 1.9 × 106M–1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrotand electron spin coherence time,Tm, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4–9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.