Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.
Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.
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Cucurbit[7]uril 增强自旋标记蛋白质的距离测量。
DOI：
10.1101/2023.08.22.554361
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Rajca,Andrzej
中科院分区：
文献类型：
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作者：
Yang,Zhimin;Stein,RichardA;Pink,Maren;Madzelan,Peter;Ngendahimana,Thacien;Rajca,Suchada;Wilson,MarkA;Eaton,SandraS;Eaton,GarethR;Mchaourab,HassaneS;Rajca,Andrzej
我们报道了氯乙酰胺2,6-二氮杂金刚烷氮氧化物自由基（ClA-DZD）与葫芦[7]uril （CB-7）之间形成的络合物，其在水中的缔合常数Ka= 1.9 × 106M-1，比先前研究的有机自由基至少高一个数量级。自由基被CB-7高度固定，表明旋转相关时间τrot比缓冲溶液中增加了36倍。ClA-DZD@CB-7的x射线结构显示未变形的CB-7宿主内封装的DZD客体，外伸出垂坠基团。将CB-7添加到T4溶菌酶（T4L）中，用DZD的碘乙酰胺衍生物进行双自旋标记，我们观察到τ旋转和电子自旋相干时间Tm增加，同时自旋间距离分布缩小。与不受CB-7影响的常见自旋标签（如MTSL）相比，DEER测量在83 K下的灵敏度增加了4-9倍。可以在水/甘油中可靠地测量3nm的旋间距离，直到接近200 K时基质的玻璃化转变/熔化温度，从而使我们更接近在接近生理温度下实现超分子识别的长距离DEER测量的目标。DZD- t4l 65在1.12 Å分辨率下的x射线结构允许DZD标签的明确建模（0.88占用），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.