Model‐Independent fluorescence polarization for measuring order in a biological assembly-猫眼课题宝

与模型无关的荧光偏振，用于测量生物组装中的顺序

DOI：

发表时间：

1984

期刊：

Biopolymers

影响因子：

2.9

通讯作者：

中科院分区：

生物学4区

文献类型：

作者：

T. Burghardt

在本文的第一部分中，我们开发了一种模型无关的方法来描述一般的、各向异性的、与时间无关的生物结构中元素排列的顺序，其中元素顺序由偶极（例如，荧光）探针。该方法首先将生物组装体的元素顺序描述为分子框架的任意角分布，其中结构的每个元素中固定有一个框架。角分布，然后展开在一个完整的正交角函数和系列扩展的时刻有关的可观测量。这些矩描述了系统的基本秩序的物理特性，而没有模型，也没有参考观察的方法。当每个元素亚基被特异性荧光标记时，该方法在此被开发用于荧光偏振测量，并且通过偏振激发后荧光探针发射的偏振来报告元素顺序。这种方法建立了荧光偏振技术的理论局限性，用于确定系统中的元素顺序，并表示分析和实验策略，以获得所有的顺序信息中观察到的信号。该方法的一个版本已成功应用于完整肌纤维中肌球蛋白跨桥排列顺序的研究[Burghardt，Ando和Borejdo（1983）Proc. Natl. Acad. Sci. USA 80，7515-7519]。本文对这些结果作了简要的讨论。在本文的第二部分中，我们将模型无关分析扩展到时间依赖的各向异性系统，其中荧光标记的元素进行受限的旋转运动。模型无关分析用于测量限制元件旋转运动的角势。该方法不需要时间分辨测量。

In the first part of this paper, we developed a model‐independent method of describing the order in the arrangement of elements in a general, anisotropic, time‐independent biologic structure, where elemental order is reported by dipolar (e.g., fluorescent) probes. This method begins with the description of the elemental order of a biologic assembly as an arbitrary angular distribution of molecular frames, with one frame fixed in each element of the structure. The angular distribution is then expanded in a complete set of orthonormal angular functions and the moments of the series expansion are related to observable quantities. These moments describe the physical properties of the elemental order of the system without a model and without reference to the method of observation. The method is developed here for fluorescence polarization measurements, when each elemental subunit is specifically fluorescence‐labeled, and the elemental order is reported by the polarization of the emission from the fluorescent probe after polarized excitation. This approach establishes the theoretical limitations of the fluorescence polarization technique for determining elemental order in a system and indicates analytical and experimental strategies for obtaining all the order information in the observed signal. A version of this method has been successfully applied to the study of the order of arrangement of the myosin cross‐bridge in intact muscle fibers [Burghardt, Ando, and Borejdo (1983) Proc. Natl. Acad. Sci. USA 80, 7515–7519]. These results are discussed briefly in this paper. In the second part of this paper we extend the model‐independent analysis to the time‐dependent, anisotropic system where the fluorescence‐labeled elements are undergoing restricted rotational motion. The model‐independent analysis is used to measure the angular potential that restricts the rotational motion of the elements. The method does not require time‐resolved measurements.

Evidence for cross-bridge order in contraction of glycerinated skeletal muscle.