Lopez Diana M, Castro Carlos E, Sotomayor Marcos
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210.
Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, Ohio 43210.
bioRxiv. 2024 Jul 22:2024.07.17.603923. doi: 10.1101/2024.07.17.603923.
Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed as tools for force measurements. Here we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide- and one DNA-based. The peptide-based sensors consist of a pair of fluorescent proteins, which can undergo Förster resonance energy transfer (FRET), linked by spider silk (GPGGA) or synthetic (GGSGGS) disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a FRET signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N- and C-terminal Cα atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand in the opposite direction. These simulations were performed at constant velocity (0.01 nm/ns - 10 nm/ns) and constant force (10 pN - 500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices.
机械力在细胞迁移和感官感知等生物过程中起着关键作用。近年来，分子力传感器已被开发为用于力测量的工具。在这里，我们使用全原子引导分子动力学模拟来预测和研究细胞生物学研究中常用的三种分子力传感器的设计参数与机械性能之间的关系：两种基于肽的和一种基于DNA的。基于肽的传感器由一对荧光蛋白组成，它们可以发生荧光共振能量转移（FRET），通过蜘蛛丝（GPGGA）或合成（GGSGGS）无序区域连接。基于DNA的传感器由两条荧光团标记的DNA链组成，在施加力时可以解开或剪切，以FRET信号作为解离的读出。我们模拟了九个传感器，每种三个。平衡后，通过在相反方向上对其N端和C端Cα原子施加力来拉伸三种不同长度的柔性肽接头。同样，我们平衡了一个基于DNA的传感器，并在相反方向上拉动一条链的末端鸟嘌呤的磷酸原子和另一条链上选定的磷酸原子。所有版本的传感器均在恒定速度（0.01 nm/ns - 10 nm/ns）和恒定力（10 pN - 500 pN）下进行这些模拟。我们的结果表明这些传感器的力响应如何取决于它们的长度、序列、构型和加载速率。从模拟分析中获得的机理见解表明，对实验结果的解释应考虑基于肽的传感器中二级结构的瞬时形成和基于DNA的传感器中过度拉伸的影响。这些预测可以指导最佳荧光团的选择，并有助于合理设计用于蛋白质、DNA、混合系统和分子装置的新型传感器。
