FLUID FORCES REGULATING PROTEIN STRUCTURE AND FUNCTION

描述(由申请人提供)： PI在血管生物学领域进行研究，重点是流体力在调节细胞和生物分子功能中的作用。他的实验室开发并应用定量实验方法来补充分子生物学和血液学的传统方法，以获得对调节白细胞-内皮细胞黏附和血栓形成机制的新见解。从长远来看，我们的目标是利用这种对生物机制和流体调节现象的理解来开发旨在治疗血管疾病的新药和治疗策略。这项工作的某些方面是由NHLBI R01拨款HL63014资助的。 在最近的实验中，我们应用我们的定量实验策略来研究剪切诱导的血小板激活。在这里，我们利用静态和动态光散射观察到，流体剪切除了调节细胞功能外，还可能调节生物分子结构。在本研究中，流体力诱导了血浆蛋白von Willebrand因子(VWF)的自关联。我们在当前的提案中更详细地研究了这一观察结果。这项建议的训练部分的目的是获得正规训练，使其能够用于研究剪切下的生物分子结构和功能。这些方法包括光散射、小角X射线和中子散射、表面等离子体共振和分子模拟。培训的某些方面要求专业人员参加研究生级别的课程和研究道德等领域的讲习班。培训的其他方面是非结构化的，它们涉及PI的自学和与合作者的互动。该项目的具体研究目标是：1)利用光、中子和X射线散射来研究剪切下血浆vWF的动态结构；2)利用表面等离子体共振来量化在模拟体内条件下vWF自结合的速率和剪切相关性；以及3)确定生物分子的结构特征，这些生物分子在生理和病理流体作用力的作用下容易发生构象变化。这是通过将上述研究获得的实验知识与分子模拟和其他计算技术中的理论方法相结合来实现的。从长远来看，这些研究旨在提供新的机制洞察力和治疗策略，以对抗心血管疾病。

DESCRIPTION (provided by applicant): The PI performs research in the area of vascular biology with emphasis on the role of fluid forces in regulating cell and biomolecule function. His laboratory develops and applies quantitative experimental methods to complement traditional methods in molecular biology and hematology in order to gain novel insight into the pathways regulating leukocyte-endothelial cell adhesion and thrombosis mechanics. In the long run, we aim to apply this understanding of biological mechanisms and fluid-flow mediated phenomena to develop new drugs and therapeutic strategies designed to treat vascular diseases. Some aspects of this work are funded by the NHLBI R01 grant HL63014. In recent experiments, we applied our quantitative experimental strategies to study shear-induced platelet activation. Here we observed using static and dynamic light scattering, that fluid shear in addition to regulating cell function, may also regulate bio-molecular structure. In this study, fluid forces induced the self-association of plasma protein von Willebrand Factor (vWF). We study this observation in greater detail in the current proposal. The objective of the training component of this proposal is to obtain formal training in methods that can be used to study biomolecule structure and function under shear. These methods include light scattering, small-angle X-ray and neutron scattering, surface plasmon resonance and molecular simulations. Some aspects of the training require the PI to attend graduate-level courses and workshops in areas including research ethics. Other aspects of training are unstructured, and they involve self-study by the PI and interactions with collaborators. The specific research goals of the project are: 1) to study the dynamic structure of plasma vWF under shear using light, neutron and X-ray scattering, 2) to quantify the rate and shear dependence of vWF self-association in a milleu that mimics in vivo conditions using surface plasmon resonance, and 3) to determine the structural features of biomolecules that make them susceptible to conformational alteration upon application of physiological and pathological fluid forces. This is achieved by combining the experimental knowledge obtained from the above studies with theoretical methods in molecular simulations and other computational techniques. In the long run, the studies aim to provide new mechanistic insight and therapeutic strategies to counter cardiovascular diseases.