Nanometric Fluorescence Imaging of Single Motor Proteins

描述(申请人提供)：肌动蛋白和非传统的肌球蛋白V和VI是负责多种亚细胞运动的分子马达。突变会导致疾病，但关于运动机制的根本问题仍然存在。比如，这些二聚体马达是以“手牵手”的方式运行，还是以“尺寸虫”的方式运行？我们已经开发出一种单分子荧光技术，可以提供答案。我们将一个荧光团连接到马达蛋白上，并以1.5纳米的空间定位、0.5秒的时间分辨率和光稳定性确定其位置，从而能够进行几分钟的观察。该方法依赖于分析发射点扩散函数(PSF)的中心，在适当的条件下，PSF代表了荧光团的位置。这项技术补充了对2-10 nm范围敏感的荧光共振能量转移和只能以纳米精度测量质心运动的光学陷阱。我们将测量这些马达蛋白在运动、空载和负载条件下的步长、角方向和头部、颈部和盘绕的柄的相对位置。 更具体地说，a)马达头部单个荧光团的单分子纳米定位将能够区分尺寸虫和手部模型(对于Kinesin，预测的步长分别为8 nm和16 nm)。使用这种技术，我们最近展示了肌球蛋白V在手到手的机制中的运动(Yildiz等人，科学，2003年)。B)我们介绍了达到单分子纳米分辨率的初步结果--即以纳米精度测量两种染料之间的距离--我们将用它来绘制电机内两个部件的相对距离和运动。例如，通过连接两个发射不同颜色的荧光团，每个头上一个荧光团，手部模型将导致交替的PSF；尺寸虫模型将导致一种颜色的PSF始终领先。C)将开发单分子定向成像，并应用于检测头、颈和茎区域的角度变化。尺寸虫模型预测茎不旋转，而对称的手部模型预测旋转，而不对称的手部模型可能没有旋转。D)通过最初用于单离子通道分析的隐马尔可夫方法对数据进行统计分析，我们还将了解是否存在与头部特定位置相对应的几个亚构象。实验将使用人类普遍存在的肌动蛋白、鸡脑肌球蛋白V和猪肌球蛋白VI-后两者采取大约36 nm的质心步骤。简要介绍了其在另一种运动--大鼠脑胞浆动力蛋白中的应用。我们预计我们的技术将适用于许多蛋白质和核酸系统。

DESCRIPTION (provided by applicant): Kinesin and the unconventional myosins V and VI are molecular motors responsible for many types of subcellular movement. Mutations lead to diseases, yet fundamental questions remain about the mechanism of motion. For example, do these dimeric motors move in a "hand-over-hand" or "inchworm" mode? We have developed a single molecule fluorescence technique that can provide answers. We attach a fluorophore to the motor protein and determine its position with 1.5 nm spatial localization, 0.5 second time resolution, and a photostability that enables observations for several minutes. The method relies on analyzing the center of the emission point-spread-function (PSF), which we have shown represents the position of the fluorophore under appropriate conditions. The technique complements fluorescence resonance energy transfer, which is sensitive to the 2-10 nm range, and optical traps, which can (only) measure center-of-mass motion (under load) with nanometer precision. We will measure the step size, angular orientation, and relative positions of the head, neck and coiled-coiled stalk of these motor proteins during motility, under no-load and loaded conditions. More specifically, A) single molecule nanometer-localization of a single fluorophore on the head of motors will be able to differentiate inchworm from hand-over-hand models (8 nm and 16 nm predicted step size, respectively, for kinesin). Using this technique, we have recently shown Myosin V moves in a hand-over-hand mechanism (Yildiz et al, Science, 2003). B) We present initial results achieving single molecule nanometer resolution -i.e., measuring the distance between two dyes with nanometer precision- which we will use to map out the relative distance and motion of two parts within the motors. For example, by attaching two fluorophores that emit different colors, one on each head, the hand-over-hand model will lead to alternating PSFs; the inchworm model will lead to the PSF of one color always leading. C) Single-molecule orientational imaging will be developed and applied to detect angular changes in the head, neck and stalk regions. An inchworm model predicts no rotation of the stalk, whereas a symmetric hand-over-hand predicts a rotation, and an asymmetric hand-over-hand model may not have a rotation. D) Through statistical analyses of data via Hidden Markov Methods, a method originally used for single ion channel analysis, we will also learn if there are several sub-conformations corresponding to a particular position of the head. Experiments will use human ubiquitous kinesin, chicken brain myosin V, and porcine myosin VI - the latter two take approximately 36 nm center-of-mass steps. Application to rat brain cytoplasmic dynein, another motor, is briefly presented. We anticipate our techniques will be applicable to many protein and nucleic acid systems.