PHOTOCONTROL OF MOTOR PROTEINS USING PHOTO-RESPONSIBLE CALMODULIN DIMER
PHOTOCONTROL OF MOTOR PROTEINS USING PHOTO-RESPONSIBLE CALMODULIN DIMER
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使用光响应钙调蛋白二聚体对运动蛋白进行光控制
DOI：
10.1016/j.bpj.2011.11.2025
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发表时间：
2012
影响因子：
3.4
通讯作者：
Hideo Seo and Shinsaku Maruta
中科院分区：
文献类型：
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作者：
Hideki Shishido;Hideo Seo and Shinsaku Maruta
钙调蛋白(CaM)是一种重要的钙离子结合蛋白，参与多种细胞调控过程。CaM在与钙离子结合后发生构象变化，使其能够与靶蛋白结合进行特异性反应。例如，钙/CaM调节肌球蛋白V、肌球蛋白轻链激酶(MLCK)等的功能。以前，我们成功地利用光致变色分子N-(4-苯基偶氮苯基)马来酰亚胺(PAM)来调控CaM的功能，PAM在紫外线(UV)和可见光(VIS)照射下发生可逆的顺反异构化。制备了在CaM功能区含有单一半胱氨酸的CaM突变体，并用PAM对其进行了修饰。在紫外光和可见光照射下，PAM-CaM与目标肽M13的结合是可逆的。在本研究中，我们将CaM突变体与双功能光致变色化合物4，4‘-偶氮苯二马来酰亚胺(ABDM)进行交联，制备了光响应性二聚体CaM，以应用于马达蛋白的光控制。对于CaM二聚体，预计每个与ABDM交联的CaM的相对特殊构型在UV-Vis光照射下会发生变化。随后，我们制备了K355-M13融合蛋白，K355-M13由运动素运动区和M13肽组成。单体K355-M13通过CaM二聚体形成二聚体构型。在有钙离子存在的情况下，CaM-ABDM-CaM与两个K355-M13结合，形成由CaM-ABDM-CaM连接的K355-M13。研究了光致变色运动蛋白二聚体的运动活性和微管依赖的ATPase活性的光调控。将CaM-ABDM-CaM应用于MLCK和肌球蛋白。
Calmodulin (CaM) is a physiologically important Ca 2+-binding protein that participates in numerous cellular regulatory processes. CaM undergoes a conformational change upon binding to Ca 2+, which enables it to bind to target proteins for specific responses. For example, Ca 2+/CaM regulates function of myosin V, myosin light chain kinase (MLCK) etc. Previously, we succeeded to photocontrol CaM function using photochromic molecule N-(4-phenylazophenyl) maleimide (PAM), which reversibly undergoes cis-trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. The CaM mutants, which have a single cysteine in the functional region of CaM, were prepared and modified with PAM. The binding of PAM-CaM to target peptide M13 was controlled reversibly upon UV and VIS light irradiation. In this study, we prepared the photo responsive dimer CaM by cross-linking of CaM mutants with bifunctional photochromic compound, 4, 4′-azobenzene-dimaleimide (ABDM) in order to apply to photo control of motor proteins. For the CaM dimer, it is expected that the relative special configuration of each of CaM cross-linked with ABDM changes by UV-VIS light irradiation. Subsequently, we prepared the fusion protein, K355-M13 composed of kinesin motor domain and M13 peptide. The monomeric K355-M13 formed dimer configuration by CaM dimer. In the presence of Ca 2+, two K355-M13 bound to the each site of CaM dimer resulted in formation of kinesin dimer linked by CaM-ABDM-CaM. The phtocontrol of the motor activity and microtubule dependent ATPase activity of the photochromic kinesin dimer was studied. Application of CaM-ABDM-CaM to MLCK and myosin was also performed.