The creation of artificial protein molecular switches

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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.