Nanometric Fluorescence Imaging of Single Motor Proteins

单运动蛋白的纳米荧光成像

• 批准号: 7072805
• 负责人: PAUL R SELVIN
• 金额: $ 29.47万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7072805
• 关键词: actins; active sites; bioimaging /biomedical imaging; cysteine; fluorescence; fluorescent dye /probe; green fluorescent proteins; ionophores; kinesin; magnetic field; method development; mutant; myosins; nanotechnology; protein localization; protein structure function; protein transport; site directed mutagenesis; statistics /biometry; three dimensional imaging /topography

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.

描述（由申请人提供）：驱动蛋白和非常规肌球蛋白 V 和 VI 是负责多种类型的亚细胞运动的分子马达。突变会导致疾病，但运动机制的基本问题仍然存在。例如，这些二聚体马达是否以“手拉手”或“尺蠖”模式移动？我们开发了一种单分子荧光技术可以提供答案。我们将荧光团附着到运动蛋白上，并以 1.5 nm 空间定位、0.5 秒时间分辨率和可观察几分钟的光稳定性来确定其位置。该方法依赖于分析发射点扩散函数（PSF）的中心，我们已经证明它代表了适当条件下荧光团的位置。该技术补充了荧光共振能量转移（对 2-10 nm 范围敏感）和光学陷阱（只能）以纳米精度测量质心运动（在负载下）。我们将测量这些运动蛋白在运动过程中、空载和负载条件下的步长、角度方向以及头部、颈部和卷曲茎的相对位置。 更具体地说，A) 电机头部单个荧光团的单分子纳米定位将能够区分尺蠖与手交模型（驱动蛋白的预测步长分别为 8 nm 和 16 nm）。使用这种技术，我们最近展示了肌球蛋白 V 以手拉手机制移动（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，通过连接两个发出不同颜色的荧光团，每个头上一个，手拉手模型将导致交替的 PSF；尺蠖模型将导致一种颜色的 PSF 始终领先。 C) 将开发并应用单分子定向成像来检测头部、颈部和柄区域的角度变化。尺蠖模型预测茎不会旋转，而对称的交手模型预测茎会旋转，而不对称的交手模型可能不会旋转。 D) 通过隐马尔可夫方法（一种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与头部的特定位置相对应的多个子构象。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 - 后两者的质心步长约为 36 nm。简要介绍了另一种马达大鼠脑细胞质动力蛋白的应用。我们预计我们的技术将适用于许多蛋白质和核酸系统。