A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin.
A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin.
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DOI：
10.1085/jgp.202012707
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发表时间：
2021-03-01
期刊：
影响因子：
--
通讯作者：
Colson BA
中科院分区：
文献类型：
--
作者：
Bunch TA;Lepak VC;Bortz KM;Colson BA
Bunch等人描述了一种新的测定法，用于快速评价溶液中cMyBP-C与F-肌动蛋白（含或不含原肌球蛋白）的结合。由于cMyBP-C磷酸化和/或HCM突变标记的肌动蛋白荧光寿命的变化与使用共沉降测定法获得的测量结果一致。肌动蛋白结合蛋白的结合特性通常通过共沉淀测定来评价。然而，这种方法很耗时，涉及多个步骤，并且吞吐量有限。这些缺点排除了其在筛选调节与人类疾病相关的肌动蛋白结合蛋白的药物中的用途。为了开发一种简单、定量和可扩展的F-肌动蛋白结合测定，我们将荧光探针连接到肌动蛋白的Cys-374上，并评估了与人心肌肌球蛋白结合蛋白C（cMyBP-C）的N-末端区域（结构域C 0-C2）结合后荧光寿命的变化。通过时间分辨荧光（TR-F）测量，所有五种测试探针的寿命在与cMyBP-C C 0-C2孵育后均降低，其中IAEDANS是产生最小误差的最灵敏探针。将TR-F测定与共沉降进行比较，以评价与肌动蛋白和肌动蛋白原肌球蛋白结合的体外变化，所述肌动蛋白和肌动蛋白原肌球蛋白由与肥厚型心肌病（HCM）和原肌球蛋白结合相关的cMyBP-C突变引起。标记的肌动蛋白与添加C 0-C2的寿命变化是一致的共沉淀结果。HCM突变L352 P被证实增强肌动蛋白结合，而PKA磷酸化降低结合。预测破坏PKA识别序列的HCM突变R282 W导致C 0-C2磷酸化缺陷和结合改变。最后，C 0-C2结合被发现是增强原肌球蛋白和结合能力被改变的原肌球蛋白结合区域的突变。这些发现表明，TR-F测定法适用于快速准确地测定定量结合，并筛选影响cMyBP-C与F-肌动蛋白结合的生理条件和化合物，用于治疗发现。
Bunch et al. describe a new assay to rapidly evaluate binding of cMyBP-C to F-actin (with or without tropomyosin) in solution. Changes in labeled actin fluorescence lifetime due to cMyBP-C phosphorylation and/or HCM mutations were consistent with measurements obtained using cosedimentation assays. Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.
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影响因子：
--
作者：
Gruber SJ;Cornea RL;Li J;Peterson KC;Schaaf TM;Gillispie GD;Dahl R;Zsebo KM;Robia SL;Thomas DD
通讯作者：
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影响因子：
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作者：
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通讯作者：
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作者：
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通讯作者：
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DOI：
10.1073/pnas.1903033116
发表时间：
2019-07-30
影响因子：
11.1
作者：
Ponnam, Saraswathi;Sevrieva, Ivanka;Kampourakis, Thomas
通讯作者：
Kampourakis, Thomas