Shahu Lalita, Gyawali Yadav Prasad, Jiang Ting, Senarathne Dedunu S, Feng Changjian, Lu H Peter
Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, United States.
Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, United States.
ACS Omega. 2025 Aug 22;10(35):39823-39832. doi: 10.1021/acsomega.5c03891. eCollection 2025 Sep 9.
Calmodulin (CaM), a calcium-sensing protein, regulates the production of nitric oxide (NO) by the nitric oxide synthase (NOS) enzyme. It is interesting to decipher the control mechanism of CaM-dependent NO biosynthesis by the NOS isoforms, as NO is a ubiquitous intercellular signaling molecule in numerous physiological processes. It is generally accepted that 4Ca-bound CaM associates with the NOS enzyme and activates NO production, while lower-level calcium has also been reported to allow for NOS enzymatic function under certain circumstances (e.g., shear stress). However, NO production by apo-CaM-activated NOS under an external force has not been directly demonstrated. Herein, we have utilized an atomic force microscopy (AFM)-correlated confocal microscopy technique to probe NO production by neuronal NOS (nNOS) enzyme, where compressive force-manipulated apo-CaM acts as a mechanosensing protein. DAR-4M was used as an NO-sensing fluorescent probe. Our results indicate that compressive force may induce necessary conformation changes of apo-CaM in binding and activating the nNOS enzyme.
钙调蛋白（CaM）是一种钙传感蛋白，可调节一氧化氮合酶（NOS）产生一氧化氮（NO）。由于NO是众多生理过程中普遍存在的细胞间信号分子，因此解读NOS同工型对CaM依赖性NO生物合成的控制机制很有趣。一般认为，与4个钙结合的CaM与NOS酶结合并激活NO的产生，不过也有报道称，在某些情况下（如剪切应力），较低水平的钙也能使NOS发挥酶功能。然而，脱钙钙调蛋白（apo-CaM）激活的NOS在外力作用下产生NO的情况尚未得到直接证实。在此，我们利用原子力显微镜（AFM）相关的共聚焦显微镜技术来探测神经元型NOS（nNOS）酶产生NO的情况，其中经压缩力操控的apo-CaM作为一种机械传感蛋白。DAR-4M用作NO传感荧光探针。我们的结果表明，压缩力可能会诱导apo-CaM在结合和激活nNOS酶时发生必要的构象变化。
