ACTIN-BASED MOTILITY BY CLAMPED-FILAMENT MOTORS

描述（由申请人提供）：我们最近介绍的动蛋白夹紧电机模型描述了如何通过夹紧丝伸长在肌动蛋白运动中产生力。我们提出了一个三步机械酶促过程（称为“锁定，加载和射击”或“LLF”机制）：在锁定过程中，一个亲和调节的夹紧蛋白（也拴在运动物体的表面）结合到位于丝的带刺端含有atp的肌动蛋白单体上；在加载步骤中，新的肌动蛋白ATP单体结合到夹紧的丝的带刺端，这一事件触发了发射步骤，在这个步骤中，夹紧的肌动蛋白亚基上的ATP水解极大地减弱了钳对丝的亲和力。ATP水解启动钳位移位并重新锁定到新的含有ATP的末端，开始另一个LLF循环。这个模型解释了表面系住的细丝是如何在运动表面施加弯曲或拉伸力时生长的，并且随机模拟再现了运动李斯特菌的特征运动。这种延伸马达利用肌动蛋白固有的atp酶活性，为力的产生提供了一个简单、高保真的酶促反应循环，而不需要将拉长的细丝从运动表面分离。我们的研究计划涉及基于模型的假设，旨在测试肌动蛋白聚合马达的LLF机制的特定特征。特异性Aim-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研究，以及(b)通过使用共价交联的profin -actin，该profin -actin在每个LLF周期后不能释放profin。特异性Aim-2侧重于(a) ATP水解在运动中的作用，使用缓慢水解ATP类似物pp(NH)pA和ATPgammaS来确定LLF机制中可能的力产生步骤。特异性Aim-3处理profilin在动态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Specific Aim-4中，我们将应用沉降和荧光各向异性测量来了解ATP水解是否以及如何调节VASP夹紧结构域与肌动蛋白丝末端的结合相互作用强度。总之，这些研究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.