Spectroscopic Probes of Smooth Muscle Functional Dynamics

描述(由申请者提供)：该项目的主要目标是为申请者提供肌肉生物化学和生物物理学方面的严格培训，为他在该领域作为一名独立研究人员的职业生涯做好准备。他在细胞生理学方面的深厚背景将通过肌肉生物物理学方面的培训而得到加强，其中特别强调收缩肌肉的电子顺磁共振，这是赞助商实验室的一个特殊优势。该培训计划的重点是一个研究项目，该项目是赞助商NIH资助的研究的核心。这个项目的总体目标是回答关于蛋白质磷酸化和动力学在平滑肌功能中的作用的基本问题，特别是强调理解具有巨大潜在生物医学重要性的平滑肌的“闩锁”状态。目的：(1)建立一种包括自旋标记和电子顺磁共振(EPR)的光谱方法，以实时准确地测量皮肤平滑肌纤维中调节轻链(RLC)的磷酸化程度。这种方法是基于赞助商实验室以前发表的文章。(2)用EPR方法定量测定肌球蛋白分子在肌动蛋白弱结合构象和强结合构象之间的分布，以及肌球蛋白分子在等长收缩时的磷酸化水平和肌力之间的关系。将特别强调肌肉进入闩锁状态的条件，并确定RLC磷酸化对这一状态的影响。(3)探讨无机磷对收缩过程的力化学动力学的扰动作用。通过完成这些目标，我们将直接测试关于平滑肌闩锁状态的分子机制的关键假说。公共卫生相关性我们正在测试有关平滑肌调节功能和失灵的基本问题。这些问题的答案将为哮喘和高血压中导致异常平滑肌收缩的潜在生物化学和生物物理学提供重要的见解。我们在收缩肌肉中的分子测量可以为药物和其他治疗方法的评估提供基础。

DESCRIPTION (provided by applicant): The primary goal of this project is to provide the applicant with rigorous training in muscle biochemistry and biophysics, to prepare him for a career as an independent investigator in this field. His strong background in cellular physiology will be augmented by training in muscle biophysics, with particular emphasis on electron paramagnetic resonance of contracting muscle, which is a particular strength of the sponsor's laboratory. This training program focuses on a research project that is central to the sponsor's NIH-funded research. The overall goal of this project is to answer fundamental questions about the role of protein phosphorylation and dynamics in the function of smooth muscle, with particular emphasis on understanding the "latch" state of smooth muscle, which has great potential biomedical importance. AIMS: (1) Develop a spectroscopic method, involving spin-labels and electron paramagnetic resonance (EPR), to measure accurately, in real time, the extent of phosphorylation of the regulatory light chain (RLC) in skinned smooth muscle fibers. This method is based closely on previous publications from the sponsor's laboratory. (2) Use this EPR method to determine quantitatively the relationship between phosphorylation level, muscle force, and the distribution of myosin molecules between weak and strong actin binding conformational states, in isometrically contracting smooth muscle. Particular emphasis will be placed on conditions under which the muscle enters the latch state, and to determine the effect of RLC phosphorylation on this state. (3) The third aim probes the effect of inorganic phosphate in perturbing the mechanochemical kinetics of the contraction process. By completing these aims, we will test directly the key hypotheses for the molecular mechanism of the latch state in smooth muscle. PUBLIC HEALTH RELEVANCE We are testing fundamental questions about the function and malfunction of smooth muscle regulation. The answers to these questions will provide crucial insights into the underlying biochemistry and biophysics responsible for abnormal smooth muscle contraction in asthma and hypertension. Our molecular measurements in contracting muscle can provide the basis for evaluation of drugs and other therapeutic approaches.