The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII autophosphorylation. The autophosphorylation in turn increases the CaMKII affinity for CAM. Here, we studied the ATP dependence of CAM association with the actin-binding CaMKIIβ isoform using single-molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface-immobilized Venus-CaMKIIβ were resolved with 0.5 s resolution from video records, batch-processed with a custom algorithm. CAM occupancy was determined simultaneously with spot-photobleaching measurement of CaMKII holoenzyme stoichiometry. We show the ATP-dependent increase of the CAM association requires dimer formation for both the α and β isoforms. The study of mutant β holoenzymes revealed that the ATP-dependent increase in CAM affinity results in two distinct states. The phosphorylation-defective (T287.306-307A) holoenzyme resides only in the low-affinity state. CAM association is further reduced in the T287A holoenzyme relative to T287.306-307A. In the absence of ATP, the affinity of CAM for the T287.306-307A mutant and the wild-type monomer are comparable. The affinity of the ATP-binding impaired (K43R) mutant is even weaker. In ATP, the K43R holoenzyme resides in the low-affinity state. The phosphomimetic mutant (T287D) resides only in a 1000-fold higher-affinity state, with mean CAM occupancy of more than half of the 14-mer holoenzyme stoichiometry in picomolar CAM. ATP promotes T287D holoenzyme disassembly but does not elevate CAM occupancy. Single Poisson distributions characterized the ATP-dependent CAM occupancy of mutant holoenzymes. In contrast, the CAM occupancy of the wild-type population had a two-state distribution with both low- and high-affinity states represented. The low-affinity state was the dominant state, a result different from published in vitro assays. Differences in assay conditions can alter the balance between activating and inhibitory autophosphorylation. Bound ATP could be sufficient for CaMKII structural function, while antagonistic autophosphorylations may tune CaMKII kinase-regulated action-potential frequency decoding in vivo.

中文翻译：

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CaMKII-钙调蛋白相互作用的实时单分子成像

钙/钙调蛋白 (CAM) 与钙/钙调蛋白依赖性蛋白激酶 II (CaMKII) 的结合启动 ATP 驱动的级联，从而触发 CaMKII 自磷酸化。自磷酸化反过来又增加了 CaMKII 对 CAM 的亲和力。在这里，我们使用单分子全内反射荧光显微镜研究了 CAM 与肌动蛋白结合 CaMKIIβ 亚型关联的 ATP 依赖性。罗丹明-CAM 与表面固定的 Venus-CaMKIIβ 的结合/解离以 0.5 秒的分辨率从视频记录中解析，并使用自定义算法进行批量处理。 CAM 占用率与 CaMKII 全酶化学计量的点光漂白测量同时测定。我们表明 CAM 关联的 ATP 依赖性增加需要 α 和 β 同工型的二聚体形成。对突变型 β 全酶的研究表明，ATP 依赖性的 CAM 亲和力增加会导致两种不同的状态。磷酸化缺陷（T287.306-307A）全酶仅处于低亲和力状态。相对于 T287.306-307A，T287A 全酶中的 CAM 关联进一步减少。在不存在 ATP 的情况下，CAM 对 T287.306-307A 突变体和野生型单体的亲和力相当。 ATP 结合受损 (K43R) 突变体的亲和力甚至更弱。在 ATP 中，K43R 全酶处于低亲和力状态。磷酸模拟突变体 (T287D) 仅处于亲和力高 1000 倍的状态，平均 CAM 占据量超过皮摩尔 CAM 中 14 聚体全酶化学计量的一半。 ATP 促进 T287D 全酶分解，但不提高 CAM 占用率。单泊松分布表征了突变全酶的 ATP 依赖性 CAM 占据。相比之下，野生型群体的 CAM 占据具有两种状态分布，即低亲和力状态和高亲和力状态。低亲和力状态是主要状态，该结果与已发表的体外测定不同。检测条件的差异可以改变激活和抑制性自磷酸化之间的平衡。结合的 ATP 可能足以实现 CaMKII 的结构功能，而拮抗性自磷酸化可能会在体内调节 CaMKII 激酶调节的动作电位频率解码。