Wyss Institute for Biologically Inspired Engineering, 3 Blackfan St., Boston, Massachusetts 02115, USA.
Chaos. 2013 Jun;23(2):025110. doi: 10.1063/1.4810915.
The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20,000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions.
可预测的基因表达调控电路的工程设计是合成生物学的一个核心目标。将启动子人工连接到转录因子基因上通常会导致噪声或混沌行为，而这种系统在实际应用中不太可能有用。天然转录调控广泛依赖于蛋白质-蛋白质相互作用来确保严格的控制，但这种严格的控制在工程系统中很难实现。为了帮助设计蛋白质-蛋白质相互作用，我们开发了一种分子动力学模拟框架，该框架简化了受约束布朗运动的蛋白质运动特征，目的是进行长时模拟。模拟蛋白质系统的行为是通过对包括布朗力、阻力、排除体积约束、相对位置约束和结合约束在内的力进行求和来确定的，这些约束与系统中选定蛋白质元件的实验确定的结合速率和离解速率有关。蛋白质被抽象为球体。结合表面在蛋白质内沿半径方向定义。肽接头被抽象为具有刚性连接的小蛋白样球体。为了确定我们的框架是否可以产生有用的预测，我们模拟了由两个 20000 Da 的蛋白质通过柔性甘氨酸/丝氨酸型接头连接而成的工程融合蛋白的行为。两个蛋白质元件紧密结合，就好像被肽接头的三维随机游动所限制一样，而不是表现出如果运动仅由蛋白质域的布朗运动主导，则预期的距离分布。我们还模拟了通过长度不同的接头连接的荧光蛋白的行为，将预测的Förster 共振能量转移与之前的实验观察结果进行了比较，并获得了很好的对应。最后，我们模拟了两个配体融合的结合行为，这两个配体可以同时结合到不同的细胞表面受体上，并探索了可以增强一个配体与受体结合的接头长度和刚度的景观，当另一个配体已经与受体结合时，从而解决了提高靶向信号转导蛋白的潜在机制。这些具体结果对涉及天然结构域融合的靶向融合蛋白和人工转录因子的设计具有重要意义。更广泛地说，这里描述的模拟框架可以扩展到包括更详细的系统特征，如非球形蛋白质形状和静电特性，而不需要详细的、计算成本高的规范。该框架应该有助于预测包括结合和解离反应在内的工程化蛋白质系统的行为。
