ACTIVE-BASED MOTILITY BY CLAMPED-FILAMENT MOTORS

通过夹紧灯丝电机实现主动运动

基本信息

批准号：
7039166
负责人：
Daniel Lee Purich
金额：
$ 21.09万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2007-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7039166
关键词：
actins adenosine triphosphate adenosinetriphosphatase biomechanics cell motility enzyme mechanism enzyme model fluorescent dye /probe hydrolysis microfilaments nucleotide analog protein protein interaction protein structure function

项目摘要

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.

描述（由申请人提供）：我们最近引入的胆红素运动模型描述了如何通过夹紧丝伸长在基于肌动蛋白的运动中产生力。我们提出了一个三步机械酶的过程（称为“锁，负载和火力”或“ LLF”机制）：在锁定期间，亲和力调节的夹具蛋白（也将其系在一起到运动型物体的表面）结合到位于灯丝杆端的含有ATP的含ATP的肌动蛋白单体上；在加载步骤中，新的肌动蛋白ATP单体结合在夹紧丝的刺齿端上，这一事件触发了射击步骤，在夹紧的肌动蛋白亚基上进行了aTP ATP水解极大地减弱了夹子的丝状亲和力。 ATP水解引发了夹具易位，并将其重新锁定到新的含ATP的末端，从而开始了另一个LLF循环。该模型解释了表面螺母的细丝如何在运动表面施加弯曲或拉伸力时如何生长，然后随机模拟再现了运动李斯特菌的标志性运动。这种伸长运动利用肌动蛋白的固有ATPase活性为力产生提供了简单的高富达酶促反应周期，不需要拉长丝从Motile表面解离。我们的研究建议介绍了基于模型的假设，旨在测试肌动蛋白聚合电动机LLF机制的特定特征。特定的AIM-1包括旨在通过在（+）结束临界浓度和（b）下进行的运动性研究来区分LLF模型与Brownian Ratchet型机制（a）的实验，并且通过使用共价交联的profilin-actin，在每个LLF周期后无法释放profilin。特定的AIM-2专注于（a）使用缓慢水解ATP类似物PP（NH）PA和ATPGAMMAS在运动中的ATP水解在运动中的作用，以鉴定LLF机制中可能产生力的步骤（S）。特定的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的负载到夹紧丝丝电机中。在特定的AIM-4中，我们将应用沉积和荧光各向异性测量值来了解ATP水解是否以及如何调节VASP夹紧域与肌动蛋白丝的末端的结合相互作用的强度。这些研究共同有望通过测试基本机械性能来开辟基于肌动蛋白的运动的新启示。