Spectroscopic Probes of Smooth Muscle Functional Dynamics

平滑肌功能动力学的光谱探针

• 批准号: 7771640
• 负责人: Joseph M. Muretta
• 金额: $ 5.15万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-25 至 2011-03-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7771640
• 关键词: ATP Hydrolysis; Actins; Address; Asthma; Binding; Biochemical; Biochemistry; Biophysics; Cell model; Cell physiology; Chemicals; Chemistry; Contracts; Coupling; Disease; Doctor of Philosophy; Drug Evaluation; Electron Spin Resonance Spectroscopy; Enzymes; Evaluation; Exhibits; Frequencies; Funding; Goals; Hypertension; Investigation; Kinetics; Label; Laboratories; Light; Measurement; Measures; Mechanics; Methods; Molecular; Molecular Conformation; Motor; Muscle; Muscle Contraction; Muscle Fibers; Myosin ATPase; Myosin Light Chain Kinase; Oryctolagus cuniculus; Phosphoric Monoester Hydrolases; Phosphorylation; Physiologic pulse; Potential Energy; Preparation; Principal Investigator; Process; Property; Protein Dephosphorylation; Proteins; Publications; Regulation; Research; Research Personnel; Research Project Grants; Resolution; Role; Skeletal Muscle; Skeletal Muscle Myosins; Skin; Smooth Muscle; Smooth Muscle Myosins; Solutions; Solvents; Speed; Spin Labels; Structural Protein; System; Techniques; Testing; Therapeutic; Time; Trachea; Training; Training Programs; Translating; Work; base; career; experience; inorganic phosphate; insight; novel therapeutic intervention; public health relevance; reproductive

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.

描述（由申请人提供）：该项目的主要目标是为申请人提供肌肉生物化学和生物物理学方面的严格培训，为他成为该领域的独立研究人员做好准备。他在细胞生理学方面的深厚背景将通过肌肉生物物理学的培训得到增强，特别强调收缩肌肉的电子顺磁共振，这是申办者实验室的一个特殊优势。该培训计划重点关注一个研究项目，该项目是赞助商 NIH 资助的研究的核心。该项目的总体目标是回答有关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，特别强调了解平滑肌的“闩锁”状态，这具有巨大的潜在生物医学重要性。目的：(1) 开发一种涉及自旋标记和电子顺磁共振 (EPR) 的光谱方法，实时准确测量带皮平滑肌纤维中调节轻链 (RLC) 的磷酸化程度。该方法紧密基于申办者实验室之前的出版物。 (2) 使用该 EPR 方法定量确定等长收缩平滑肌中磷酸化水平、肌肉力量以及弱肌动蛋白结合构象状态和强肌动蛋白结合构象状态之间的肌球蛋白分子分布之间的关系。将特别强调肌肉进入闩锁状态的条件，并确定 RLC 磷酸化对此状态的影响。 (3)第三个目标探讨无机磷酸盐在扰动收缩过程的机械化学动力学中的作用。通过完成这些目标，我们将直接测试平滑肌闩锁状态分子机制的关键假设。公共卫生相关性 我们正在测试有关平滑肌调节功能和故障的基本问题。这些问题的答案将为了解哮喘和高血压中平滑肌收缩异常的潜在生物化学和生物物理学提供重要的见解。我们对肌肉收缩的分子测量可以为药物和其他治疗方法的评估提供基础。

项目成果

A continuous fluorescence displacement assay for BioA: an enzyme involved in biotin biosynthesis.

• DOI: 10.1016/j.ab.2011.05.003
• 发表时间: 2011-09-01
• 期刊: Analytical biochemistry
• 影响因子: 2.9
• 作者: Wilson DJ;Shi C;Duckworth BP;Muretta JM;Manjunatha U;Sham YY;Thomas DD;Aldrich CC
• 通讯作者: Aldrich CC