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Probing weak single-molecule interactions: Development and demonstration of a new instrument.

机译：探测弱的单分子相互作用：新仪器的开发和演示。

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摘要

The focus of this thesis is the development, verification, and scientific use of a force probe designed to explore weak interactions between or within single biomolecules. The developed system utilizes a laser optical trap to apply piconewton forces to a functionalized probe bead that interacts with a reactive test bead, and high-speed video processing to measure nanometer displacements of both beads at a rate of ∼1500 Hz. The position of the probe bead is used to report both the force on a single molecular bond and the change in length due to protein unfolding or refolding. Several feedback and automation systems have been integrated to facilitate the repetitive testing required to characterize these stochastic interactions.; The measurement and application of piconewton-sized forces depends on accurate position detection and proper calibration of the optical trap. The accuracy of the position detection was determined to be 2-3 nm by analyzing a non-moving bead. To assess optical trap calibration, a detailed study and comparison of common methods was performed. Noise-based methods which rely on the variance or power spectrum were found to have systematic errors due to the finite integration time of the detection. To solve this problem, the effect of integration time on these measurements was quantified and new calibration methods were developed.; Finally, the instrument was used to study the forced unfolding of the spectrin repeat, a small modular protein domain found in various load supporting proteins. The unfolding kinetics were determined by pulling on the molecule over several orders of magnitude in loading rate. The findings indicate that spectrin unfolding is governed by a single prominent energy barrier, characterized by a force scale of 2.5 pN and a stress-free unfolding rate of 0.003 s -1.
机译：本文的重点是力探针的开发，验证和科学使用，该探针旨在探索单个生物分子之间或内部的弱相互作用。开发的系统利用激光陷阱将微微网力施加到与反应性测试珠相互作用的功能化探针珠上，并利用高速视频处理以〜1500 Hz的速率测量两个珠的纳米位移。探针珠的位置既可用于报告单个分子键上的力，也可用于报告由于蛋白质解折叠或重折叠而引起的长度变化。集成了多个反馈和自动化系统，以促进表征这些随机相互作用所需的重复测试。皮克顿力的测量和应用取决于准确的位置检测和对光阱的正确校准。通过分析不动的珠子，位置检测的精度确定为2-3 nm。为了评估光阱校准，进行了详细的研究并比较了常用方法。由于检测的有限积分时间，依赖于方差或功率谱的基于噪声的方法被发现具有系统误差。为了解决这个问题，量化了积分时间对这些测量的影响，并开发了新的校准方法。最后，该仪器用于研究血影蛋白重复序列​​的强制展开，血影蛋白重复序列​​是在各种负载支持蛋白中发现的小模块化蛋白结构域。展开动力学是通过将分子拉至超过几个数量级的负载速率来确定的。这些发现表明，血影蛋白的展开受单个突出的能垒控制，其特征是力规模为2.5 pN，无应力展开速率为0.003 s -1。

著录项

  • 作者

    Halvorsen, Kenneth Anders.;

  • 作者单位

    Boston University.;

  • 授予单位 Boston University.;
  • 学科 Engineering Biomedical.; Biophysics General.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 148 p.
  • 总页数 148
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物医学工程;生物物理学;
  • 关键词

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