Department of Bioengineering, University of Washington, Seattle, Washington, USA; Center for Translational Muscle Research, University of Washington, Seattle, Washington, USA.
Center for Translational Muscle Research, University of Washington, Seattle, Washington, USA; Division of Cardiology, University of Washington, Seattle, Washington, USA.
J Biol Chem. 2024 Jan;300(1):105565. doi: 10.1016/j.jbc.2023.105565. Epub 2023 Dec 14.
The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (K, 0.32 and 1.76 μM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.
肌球蛋白的生化 SRX（超级松弛）状态被定义为低 ATPase 活性状态。当肌球蛋白未被招募用于肌肉收缩时，这种状态可以节省能量。SRX 状态与肌球蛋白厚丝的结构定义的有序（相对于无序）状态相关。这两种状态可能通过一个共同的相互作用头部模体（IHM）相关联，其中重酶解肌球蛋白（HMM）或肌球蛋白的两个头部折叠在一起，并与 S2 和厚丝形成额外的接触。然而，对 SRX、IHM 和厚丝有序形式的实验观察并不总是一致的，并且导致了一系列未解决的悖论。为了解决这些悖论，我们重新检查了猪心 HMM 的 SRX 状态的生化测量。在我们的手中，通常采用的 mantATP 置换测定法无法定量 SRX 状态的群体，所有数据都非常好地拟合单指数。我们进一步表明，mavacamten 抑制了猪心室 HMM 和 S1 的基础 ATPases（分别为 K，0.32 和 1.76 μM），而 dATP 协同激活 HMM，没有任何 SRX 状态的证据。我们的实验观察和理论的结合表明，在纯化蛋白中 mantATP 的置换不是定量 SRX 群体的可靠测定法。这意味着，虽然结构定义的 IHM 和有序的厚丝显然存在，但在使用 mantATP 置换测定法时必须非常小心。
