Rotation of Single Cross-Bridge in Skeletal Muscle Fiber

骨骼肌纤维中单个横桥的旋转

• 批准号: 7256347
• 负责人: JULIAN BOREJDO
• 金额: $ 14.96万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7256347
• 关键词: Active Sites; Anisotropy; Binding; Catalytic Domain; Characteristics; Chemicals; Contracts; Coupled; Development; Dissociation; Event; Fluorescence; Fluorescence Anisotropy; Generations; Individual; Isometric Contraction; Kinetics; Light; Lighting; Measurement; Measures; Mechanics; Methods; Microscope; Monitor; Movement; Muscle; Muscle Contraction; Muscle Fibers; Myosin ATPase; Myosin S-1; Numbers; Phosphate-Binding Proteins; Photobleaching; Photons; Population; Positioning Attribute; Rate; Rotation; Signal Transduction; Site; Skeletal Muscle; Spectrum Analysis; Testing; Time; Work; inorganic phosphate; research study

DESCRIPTION (provided by applicant): The current hypothesis of contraction of skeletal muscle is that the binding of a "fuel" molecule (ATP) to an active site of myosin induces a local conformational change in the catalytic domain --an enzymatically active part of a molecule. This change is mechanically amplified and leads to a major rotation of the regulatory domain -- a long part at the end of myosin that is enzymatically inert. The rotation of the regulatory domain results in the generation of force and movement. The rotation is coupled to the chemical events occurring at the active site of myosin. The aim of this proposal is to test this hypothesis in a single cross-bridge of contracting muscle fiber. A confocal microscope is modified to allow measurements from a small population (approximately 10) of cross-bridges. The rotation of the regulatory domain and the enzymatic activity are measured simultaneously. The rotation is studied by measuring the anisotropy of fluorescence of probes placed at strategic positions within the regulatory domain. The anisotropy is measured either during transient contraction (created by suddenly releasing ATP from a cage) or during steady-state contraction (by using correlation spectroscopy method). The enzymatic activity is measured by fluorescence of phosphate binding protein excited by light emerging from a Near-Field probe. The anisotropy and enzymatic signals are cross-correlated to establish their causal relationship. The significance of this work is that the prevailing hypothesis will be tested, for the first time in a single cross-bridge of working muscle. This is expected to provide definitive answers about the mechanism of contraction of skeletal muscle.

描述（由申请人提供）：目前骨骼肌收缩的假设是“燃料”分子（ATP）与肌球蛋白活性位点的结合诱导催化结构域（肌球蛋白的酶活性部分）的局部构象变化。一个分子。这种变化被机械放大并导致调节域的重大旋转——肌球蛋白末端的长部分，具有酶惰性。调节域的旋转导致力和运动的产生。旋转与肌球蛋白活性位点发生的化学事件相关。该提案的目的是在收缩肌纤维的单个横桥中检验这一假设。共焦显微镜经过改进，可以对一小部分（大约 10 个）跨桥进行测量。同时测量调节域的旋转和酶活性。通过测量放置在调节域内战略位置的探针的荧光各向异性来研究旋转。各向异性可以在瞬时收缩期间（通过从笼中突然释放 ATP 产生）或在稳态收缩期间（通过使用相关光谱法）进行测量。通过由近场探针发出的光激发的磷酸盐结合蛋白的荧光来测量酶活性。各向异性和酶信号相互关联以建立它们的因果关系。这项工作的意义在于，将首次在单一的工作肌肉跨桥中检验普遍的假设。预计这将为骨骼肌收缩机制提供明确的答案。

项目成果

Fluorescence correlation spectroscopy in a reverse Kretchmann surface plasmon assisted microscope.

• DOI: 10.1364/oe.16.013381
• 发表时间: 2008-08
• 期刊: Optics express
• 影响因子: 3.8
• 作者: N. Calander;P. Muthu;Z. Gryczynski;I. Gryczynski;J. Borejdo

Rotations of a few cross-bridges in muscle by confocal total internal reflection microscopy.

通过共焦全内反射显微镜观察肌肉中一些横桥的旋转。

• DOI: 10.1016/j.bbamcr.2005.11.011
• 发表时间: 2006
• 期刊: Biochimica et biophysica acta
• 作者: Borejdo,J;Talent,J;Akopova,I;Burghardt,TP
• 通讯作者: Burghardt,TP

Cross-bridge duty cycle in isometric contraction of skeletal myofibrils.

骨骼肌原纤维等长收缩的跨桥占空比。

• DOI: 10.1021/bi7023223
• 发表时间: 2008
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Muthu,P;Talent,JM;Gryczynski,I;Borejdo,J
• 通讯作者: Borejdo,J

Simultaneous measurement of rotations of myosin, actin and ADP in a contracting skeletal muscle fiber.

同时测量收缩骨骼肌纤维中肌球蛋白、肌动蛋白和 ADP 的旋转。

• DOI: 10.1007/s10974-004-5073-6
• 发表时间: 2004
• 期刊: Journal of muscle research and cell motility
• 影响因子: 2.7
• 作者: Shepard,AA;Dumka,D;Akopova,I;Talent,J;Borejdo,J
• 通讯作者: Borejdo,J

A Novel Method of Determining the Functional Effects of a Minor Genetic Modification of a Protein.

• DOI: 10.3389/fcvm.2015.00035
• 发表时间: 2015
• 期刊: Frontiers in cardiovascular medicine
• 影响因子: 3.6
• 作者: Nagwekar J;Duggal D;Midde K;Rich R;Liang J;Kazmierczak K;Huang W;Fudala R;Gryczynski I;Gryczynski Z;Szczesna-Cordary D;Borejdo J
• 通讯作者: Borejdo J