ACTIN-BASED MOTILITY BY CLAMPED-FILAMENT MOTORS

描述（由申请人提供）：我们最近推出的actoclampin电机模型描述了如何通过夹紧丝伸长在肌动蛋白为基础的运动力。我们提出了一个三步机械酶过程（称为“锁定，加载和发射”或“LLF”机制）：在锁定过程中，亲和力调节的钳蛋白（也被拴在运动物体的表面）结合到位于细丝的倒刺末端的含ATP的肌动蛋白单体上;在加载步骤中，新的肌动蛋白ATP单体结合到夹紧细丝的倒刺末端上，这是触发发射步骤的事件，因此，在被夹的肌动蛋白亚基上的ATP水解大大减弱了夹对细丝的亲和力。ATP水解启动钳易位并重新锁定到新的含ATP的末端，开始另一个LLF循环。这个模型解释了表面束缚的细丝如何在运动表面上施加弯曲或张力的同时生长，随机模拟再现了运动李斯特菌的签名运动。这种伸长马达利用肌动蛋白的内在ATP酶活性来提供用于力产生的简单、高保真的酶促反应循环，其不需要伸长的细丝从运动表面解离。我们的研究建议地址基于模型的假设，旨在测试肌动蛋白聚合马达的LLF机制的具体功能。具体目标-1包括旨在区分LLF模型与布朗棘轮型机制的实验，（a）通过在（+）端临界浓度附近和以下进行运动性研究，和（B）通过使用在每个LLF循环后不能释放profilin的共价交联的profilin-actin。具体目标-2侧重于（a）ATP水解在运动中的作用，使用缓慢水解的ATP类似物pp（NH）pA和ATP γ S来鉴定LLF机制中可能的力产生步骤。Specific Aim-3涉及profilin在动力学校对途径中的潜在作用，以抑制肌动蛋白-ADP加载到夹丝马达中。在具体目标-4，我们将应用沉降和荧光各向异性测量，以了解ATP水解是否以及如何调节VASP的夹紧结构域与肌动蛋白丝末端的结合相互作用的强度。总之，这些调查有望通过测试基本的机械特性来揭示基于肌动蛋白的运动性。

DESCRIPTION (provided by applicant): Our recently introduced actoclampin motor model describes how force is generated in actin-based motility through clamped-filament elongation. We proposed a three-step mechanoenzymatic process (called the "Lock, Load & Fire" or "LLF" mechanism): during locking, an affinity-modulated clamp protein (also tethered to the surface of a motile object) binds onto ATP-containing actin monomers situated at a filament's barbed-end; in the loading step, new actin ATP monomers bind onto the barbed end of a clamped-filament, an event that triggers the firing step, whereto ATP hydrolysis on the clamped actin subunit greatly attenuates the clamp's affinity for the filament. ATP hydrolysis initiates clamp translocation and re-locking to the new ATP-containing terminus, starting another LLF cycle. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface, and stochastic simulations reproduce the signature motions of motile Listeria. This elongation motor exploits actin's intrinsic ATPase activity to provide a simple, high fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. Our research proposal addresses model-based hypotheses designed to test specific features of the LLF mechanism of actin polymerization motors. Specific Aim-1 includes experiments aimed at differentiating the LLF model from Brownian Ratchet-type mechanisms (a) by conducting motility studies near and below the (+)-end critical concentration, and (b) by using covalently cross-linked profilin-actin that cannot release profilin after each LLF cycle. Specific Aim-2 focuses on (a) the role of ATP hydrolysis in motility using slowly hydrolyzing ATP analogues pp(NH)pA and ATPgammaS to identify the likely force-producing step(s) in the LLF mechanism. Specific Aim-3 deals with profilin's potential role in a kinetic proofreading pathway to suppress loading of actin-ADP into clamped-filament motors. In Specific Aim-4, we will apply sedimentation and fluorescence anisotropy measurements to learn if and how ATP hydrolysis modulates the strength of binding interactions of VASP's clamping domain with the ends of actin filaments. Together, these investigations promise to shed new light on actin-based motility by testing fundamental mechanistic properties.