用光镊测定单分子水平的蛋白质-蛋白质相互作用。

IF 7.2 2区 生物学 Q1 BIOPHYSICS
Wendy N Sánchez, Luka Robeson, Valentina Carrasco, Nataniel L Figueroa, Francesca Burgos-Bravo, Christian A M Wilson, Nathalie Casanova-Morales
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引用次数: 3

摘要

生物分子相互作用是生物体内所有物理过程的基础;对这些相互作用的研究导致了大量不同方法的发展。其中,单分子实验近年来变得相关,因为这些研究可以深入了解隐藏在基于集成(多)方法中的机制和相互作用。本文综述了光学镊子实验,该实验可用于应用和测量分子系统中的机械力。OTs是基于光捕获,其中激光被用来对介电珠施加一个力;并在三维空间中以光学方式将头部锁定在可控位置。已经开发了不同的实验方法来研究使用OTs的蛋白质-蛋白质相互作用,例如:(1)反式相互作用中的重折叠和展开,其中一个蛋白质被拴在珠子之间,另一个蛋白质在溶液中;(2)在顺式相互作用中,当每个蛋白质与一个头结合时,力恒定,张力突然增加。相互作用可能在一段时间后中断,从而提供有关该张力下结合寿命的信息。(3)顺式相互作用中的斜坡力,每个蛋白质附着在一个头上,施加斜坡力,直到相互作用破裂。通过这些实验,可以得到动力学常数(koff, kon)、亲和值(KD)、到过渡态的能量ΔG≠、到过渡态的距离Δx≠等参数。这些参数表征了相互作用的能量格局。一些参数,如到过渡态的距离,只能从力谱实验中获得,比如这里描述的那些。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Determination of protein-protein interactions at the single-molecule level using optical tweezers.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein–protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (koff, kon), affinity values (KD), energy to the transition state ΔG, distance to the transition state Δx can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

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来源期刊
Quarterly Reviews of Biophysics
Quarterly Reviews of Biophysics 生物-生物物理
CiteScore
12.90
自引率
1.60%
发文量
16
期刊介绍: Quarterly Reviews of Biophysics covers the field of experimental and computational biophysics. Experimental biophysics span across different physics-based measurements such as optical microscopy, super-resolution imaging, electron microscopy, X-ray and neutron diffraction, spectroscopy, calorimetry, thermodynamics and their integrated uses. Computational biophysics includes theory, simulations, bioinformatics and system analysis. These biophysical methodologies are used to discover the structure, function and physiology of biological systems in varying complexities from cells, organelles, membranes, protein-nucleic acid complexes, molecular machines to molecules. The majority of reviews published are invited from authors who have made significant contributions to the field, who give critical, readable and sometimes controversial accounts of recent progress and problems in their specialty. The journal has long-standing, worldwide reputation, demonstrated by its high ranking in the ISI Science Citation Index, as a forum for general and specialized communication between biophysicists working in different areas. Thematic issues are occasionally published.
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