Building molecular transport machines regulated by autoinhibition and ligand-induced activatio

所有的生命都利用分子机器。这些可以被放在许多复杂的任务中，包括使细胞保持和调整其形状，移动组件，以及分裂形成新的细胞。在这里，我们将解决合成生物学日益增长的领域中的两个关键挑战：我们如何建造分子机器？那么，我们如何将它们整合到自然系统中呢？随着我们设计生物学以获得对自然生物系统的基本了解，以及我们如何在未来将这些知识转化为新兴工程生物学领域的生物技术应用，这些问题的答案将有许多未来的应用。分子机器的活动必须受到严格控制。我们研究了这种控制是如何在自然系统中实现的。通常，当机器的一个部分与另一个部分相互作用时，就会发生这种情况--本质上是卡住了机械装置。这种堵塞可以在特定的提示下解除阻止，从而允许触发活动。这种干扰现象被称为“自身抑制”，而这种提示被称为“配体诱导激活”。我们想学习如何构建自我调节的分子机器，这些机器可以通过合理的蛋白质设计和工程引入特定的提示来激活。我们受到了天然运输机器--细胞骨架马达--的启发，这些机器经常使用这种机制。我们将专注于我们拥有特殊专业知识的微管马达的Kinesin家族。为此，我们将从自然系统中提取组件，并将它们与我们将重新设计或从头设计的组件结合在一起。这些将包括Kinesin ATPase运动域(A)、来自Woolfson实验室的从头设计的螺旋线圈(B)、用于货物识别和自身抑制的设计的鞑靼重复蛋白(dArmRPs，C)，以及用于细胞成像的荧光团(D)。关键的挑战将是让这些组件在细胞中协同工作。这将需要对天然组件和设计组件之间的相互作用进行微调。我们将继续使用该系统来整合复杂的调控机制，如磷酸化，并询问我们如何结合相反方向的电机或利用其他细胞骨架轨迹，如肌球蛋白和肌动蛋白。

All life exploits molecular machines. These can be put to a multitude of complex tasks that include enabling cells to maintain and adapt their shape, move components around, and divide to make new cells. Here, we will address two key challenges in the growing field of synthetic biology:How do we build molecular machines? And, how do we integrate them into natural systems? The answers to these questions will have many future applications as we engineer biology to obtain fundamental understanding of natural biological systems, and how we translate this knowledge in the future for biotechnology applications in the emerging area of engineering biology.The activities of molecular machines have to be tightly controlled. We have studied how this control is achieved in natural systems. Often, this occurs when one part of the machine interacts with another part - essentially jamming the mechanism. This jam can be unblocked on specific cues, and so allows activity to be triggered. This jamming phenomenon is known as 'autoinhibition' and the cue is known as 'ligand-induced activation'. We want to learn how to build self-regulated molecular machines that can be activated by specific cues introduced through rational protein design and engineering. We are inspired by natural transport machines - the cytoskeletal motors - which often use such mechanisms. We will focus on the kinesin family of microtubule motors where we have particular expertise.To do this, we will take component parts from natural systems and combine them with parts that we will design de novo or from scratch. These will include kinesin ATPase motor domains (A), de novo designed coiled coils from the Woolfson lab (B), designed armadillo repeat proteins (dArmRPs, C) for cargo recognition and autoinhibiton, and fluorophores for cell imaging (D). The keychallenge will be making these components work together in cells. This will require fine tuning of interactions between the natural and designed components. We will move on to use the system to incorporate sophisticated regulatory mechanisms such as phosphorylation, and ask how we can combine motors of opposite directionality or that utilise other cytoskeletal tracks such as myosins and actin.