The creation of artificial protein molecular switches

生物体感知环境变化并以适当方式作出反应的能力对其生存至关重要。细胞必须对许多刺激做出反应，包括化学信号，如营养水平和信使分子的变化。因此，自然界已经进化出许多不同的系统，能够对信号做出反应。一般来说，蛋白质通过识别和结合负责的化学物质来充当刺激的传感器。在结合化学物质时，蛋白质的三维结构发生变化，从而改变其功能。正是这种功能上的变化，使信号被检测到，并通过激活链中的下一个环节或允许蛋白质直接解决刺激的原因而做出反应。这种识别和结合化学信号导致其结构和功能变化的蛋白质被称为蛋白质分子开关。产生能够响应于所需输入而改变其性质（或输出）的分子的能力具有广泛的应用潜力，包括创建新型传感器和材料。由于已知蛋白质具有适合于分子开关的性质，因此它们将是构建我们所需开关的理想材料，这似乎是合乎逻辑的。虽然使用天然传感蛋白可能看起来最简单，但这些蛋白已经进化为在定义的生物过程中实现特定功能，并且可能不具有特定应用所需的特性。因此，可能需要利用和适应新的蛋白质。蛋白质结构的天然多样性表明蛋白质是非常灵活的分子，能够发挥广泛的功能。我们通过修改编码蛋白质的DNA序列来增加这种多样性的能力，也扩大了对蛋白质开放的结构排列的多样性，从而允许新的特性被纳入。产生蛋白质，其输出响应于所需的化学物质而变化，将为感知细胞环境的变化并引发适当的反应提供有力的工具。人工蛋白质开关也可以在生物学背景之外应用，例如在纳米技术领域。由于蛋白质在纳米尺度上工作，它们作为分子开关的能力可以应用于创造新的传感器，换能器和智能材料，以快速和可逆的方式做出反应。因此，我们假设可以创造人工蛋白质，它们可以充当由选择输入控制的分子开关。拟议的研究将通过创建一种新的分子开关来解决这一假设，该开关对生物学上重要的小分子血红素做出反应。为了实现这一目标，我们将血红素与细胞色素b562（cyt B）蛋白结合时发生的结构变化与TEM-1 β-内酰胺酶的催化活性联系起来。Cyt B和TEM-1在自然界中具有不相关的功能，并且作为单独的蛋白质存在。为了连接它们的功能，将使用称为结构域插入的策略，其中一个蛋白质插入另一个蛋白质中。在这种情况下，将使用最近开发的遗传工程工具将cyt B插入TEM-1中。由于难以预测蛋白质内允许插入另一个蛋白质同时保留单个蛋白质的功能并允许血红素结合事件与酶活性偶联的位点，因此将对TEM-1内的许多不同插入位置进行采样。新的嵌合蛋白将被分析，以确定和分离那些TEM-1活性现在依赖于血红素结合。将更详细地分析具有所需开关属性的那些嵌合蛋白质以验证它们的性质。

The ability of an organism to sense changes to its environment and react in a suitable manner is critical to its survival. Cells must respond to many stimuli, including chemical signals such as changes in nutrient levels and messenger molecules. Consequently, nature has evolved many different systems capable of responding to the signal. Generally, proteins act as the sensor of the stimulus by recognising and binding the chemicals responsible. On binding the chemical, the three-dimensional structure of the protein changes so altering its function. It is this change in function that allows the signal to be detected and reacted upon by activating the next link in the chain or allowing the protein to directly tackle the cause to the stimulus. Such proteins that recognise and bind chemical signals leading to a change in their structure and function are termed protein molecular switches. The ability to produce molecules that can change their properties (or output) in response to a desired input has potential for a wide variety of applications, including the creation of novel sensors and materials. As proteins are already known to have the properties suitable for a molecular switch, it would appear logical that they would be the ideal material from which to construct our desired switches. Although it might appear simplest to use natural sensing proteins, these have evolved to fulfil specific functions within a defined biological process and may not have the requisite properties for a particular application. Therefore, new proteins may need to be utilised and adapted. The natural diversity of protein structure illustrates that proteins are very flexible molecules capable of a wide range of functions. Our ability to increase this diversity by modifying the DNA sequence that encodes a protein, also broadens the variety of structural permutations that are open to proteins thus allowing new properties to be incorporated. The creation of proteins whose output changes in response to a desired chemical will provide a powerful tool for sensing changes in the cellular environment and eliciting a suitable response. Artificial protein switches could also have applications outside of the biological context, such as in the area of nanotechnology. As proteins work at the nanometre scale, their ability to act as a molecular switch could be applied to create novel sensors, transducers and intelligent materials that respond in a required manner rapidly and reversibly. Therefore, we hypothesise that artificial proteins can be created that can act as molecular switches controlled by an input of choice. The proposed research will address this hypothesis by creating a novel molecular switch that responds to the biologically important small molecule haem. To achieve this, we will link the structural changes that occur on haem binding to the protein cytochrome b562 (cyt b) to the catalytic activity of the enzyme TEM-1 beta-lactamase. Cyt b and TEM-1 have unrelated functions in nature and exist as separate proteins. In order to link their functions, a strategy called domain insertion will be used, in which one protein is inserted within another. In this case, cyt b will be inserted within TEM-1 using a recently developed genetic engineering tool. As it is difficult to predict sites within a protein that permit the insertion of another while retaining the functions of the individual proteins and allowing events on haem binding to be coupled to enzyme activity, many different insertion positions within TEM-1 will be sampled. The new chimeric proteins will be analysed to identify and isolate those whose TEM-1 activity is now dependent on haem binding. Those chimeric proteins with the desired switching attributes will be analysed in more detail to characterise their properties.