Mas Guillaume, Hiller Sebastian
Biozentrum, University of Basel, Basel, Switzerland.
Nat Commun. 2025 Jun 1;16(1):5086. doi: 10.1038/s41467-025-60343-x.
The 70 kDa heat shock protein (Hsp70) family of molecular chaperones ensures protein biogenesis and homeostasis, driven by ATP hydrolysis. Here, we introduce in-cyclo NMR, an experimental setup that combines high-resolution NMR spectroscopy with an ATP recovery and a phosphate removal system. In-cyclo NMR simultaneously resolves kinetic rates and structural information along functional cycles of ATP-driven molecular machines. We benchmark the method on the nucleotide binding domain (NBD) of the human Hsp70 chaperone BiP. The protein cycles through ATP binding, hydrolysis, and two parallel pathways of product release. We determine the kinetic rates of all eleven underlying elementary reactions and show these to match independent measurements. The two product release pathways regulate the cycle duration dependent on the products concentration. Under physiological conditions, they are both used. The in-cyclo NMR method will serve as a platform for studies of ATP-driven functional cycles at a remarkable level of detail.
分子伴侣的70 kDa热休克蛋白（Hsp70）家族通过ATP水解驱动，确保蛋白质生物合成和稳态。在此，我们引入了环内核磁共振技术，这是一种将高分辨率核磁共振光谱与ATP回收和磷酸盐去除系统相结合的实验装置。环内核磁共振技术能够同时解析ATP驱动分子机器功能循环中的动力学速率和结构信息。我们以人类Hsp70伴侣BiP的核苷酸结合结构域（NBD）为该方法的基准进行测试。该蛋白质经历ATP结合、水解以及产物释放的两条平行途径的循环。我们确定了所有11个潜在基本反应的动力学速率，并表明这些速率与独立测量结果相符。两条产物释放途径根据产物浓度调节循环持续时间。在生理条件下，两条途径都会被使用。环内核磁共振技术将作为一个平台，用于在显著的细节水平上研究ATP驱动的功能循环。
