Nanometric Fluorescence Imaging of Single Motor Proteins

描述（由申请人提供）：驱动蛋白和非常规肌球蛋白V和VI是负责多种类型亚细胞运动的分子马达。突变导致疾病，但运动机制的基本问题仍然存在。例如，这些二聚体马达是以“双手交替”还是“尺蠖”模式移动？我们已经开发出一种单分子荧光技术，可以提供答案。我们将荧光团连接到马达蛋白，并以1.5 nm的空间定位，0.5秒的时间分辨率和能够观察几分钟的光稳定性确定其位置。该方法依赖于分析发射点扩散函数（PSF）的中心，我们已经表明，在适当的条件下，它代表了荧光团的位置。该技术补充了对2-10 nm范围敏感的荧光共振能量转移，以及可以（仅）以纳米精度测量质心运动（负载下）的光学陷阱。我们将测量的步长，角取向，和相对位置的头部，颈部和卷曲螺旋柄的运动过程中，在空载和负载条件下，这些马达蛋白。 更具体地，A）单个荧光团在马达头部上的单分子纳米定位将能够区分尺蠖与手交模型（对于驱动蛋白，预测步长分别为8 nm和16 nm）。使用这种技术，我们最近已经显示肌球蛋白V以双手交替的机制移动（Yildiz等人，Science，2003）。B）我们提出了实现单分子纳米分辨率的初步结果-即，以纳米级的精度测量两种染料之间的距离-我们将用它来绘制出马达内两个部件的相对距离和运动。例如，通过附加两个发出不同颜色的荧光团，每个头上一个，双手交替模型将导致交替的PSF;尺蠖模型将导致一种颜色的PSF总是领先。C）将开发和应用单分子定向成像来检测头部、颈部和柄部区域的角度变化。尺蠖模型预测茎没有旋转，而对称的手对手模型预测旋转，并且不对称的手对手模型可能没有旋转。D）通过隐马尔可夫方法（最初用于单离子通道分析的方法）对数据进行统计分析，我们还将了解是否存在对应于头部特定位置的几个子构象。实验将使用人类普遍存在的驱动蛋白，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。应用大鼠脑细胞质动力蛋白，另一个电机，简要介绍。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.