The creation of artificial protein molecular switches

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences

Abstract

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.

Technical Summary

The ability to design molecules that can change their properties in response to a desired input will allow significant new possibilities for creating novel sensors, modulators and transducers for use in both natural and artificial contexts. The capacity of proteins to fulfil the function of a molecular switch is well established in nature, as demonstrated by their key roles in regulating many biological processes. The aim of this project is to create artificial protein molecular switches whose output is modulated by an input of choice. While it might appear easiest to modify natural protein switches, they have evolved to fulfil specific requirements within a certain biological context, making them difficult to adapt for new applications. Therefore, as a general approach for creating tailored protein switches, we hypothesise that unrelated proteins with the requisite properties can be coupled through ligand-dependent conformational events. To address this hypothesis, the protein TEM-1 beta-lactamase will be converted into an allosteric enzyme whose activity is regulated by the biologically important small molecule haem. A strategy called domain insertion will be used to link the enzyme activity of TEM-1 with the conformational changes associated with haem binding to cytochrome b562 (cyt b). As it is difficult to predict sites within a protein that will permit the insertion of another protein while retaining the function of both proteins and allowing the transmission of conformational events, a directed evolution approach will be used. Libraries of chimeric proteins will be created using a recently developed transposon method in which cyt b will be inserted randomly into TEM-1. The libraries will be screened to identify chimeric proteins whose TEM-1 activity is dependent on haem binding. Those chimeras with the desired switching properties will be analysed in more detail in vitro and in vivo to characterise their haem dependent properties.

Publications

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Della Pia EA (2012) Direct binding of a redox protein for single-molecule electron transfer measurements. in Small (Weinheim an der Bergstrasse, Germany)

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Della Pia EA (2012) Redox tuning of cytochrome b562 through facile metal porphyrin substitution. in Chemical communications (Cambridge, England)

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Jones DD (2014) Transposon-based approaches for generating novel molecular diversity during directed evolution. in Methods in molecular biology (Clifton, N.J.)

 
Description 1. Development and demonstration of a general protein mutagenesis technology. The underlying whole gene random domain insertion approach was successfully implemented and extended to sample different mutational events during the course of the grant.

2. The construction of novel proteins not present in nature that can act as molecular switches both in vitro and in vivo, and molecular insights into how the switching process occurs.

3. Determination of the first (to our knowledge) molecular structure of an engineered "domain insert protein".

4. The demonstration that domain insertion is a valid strategy for generating molecular switches. This has in turn led to this approach being applied to different systems and applications, including synthetic biology.
Exploitation Route The ability to create artificial protein molecular switches with properties desirable to the researcher will prove an invaluable tool for the biological sciences. Such proteins will allow the researcher to control molecular and cellular events by a chosen signal, helping to investigate the roles played by various proteins and pathways in various biological processes. It will also allow the researcher to create novel sensors that can detect changes in cellular conditions and elicit a suitable response. Researchers will also be able to create protein switches that act as novel regulatory elements in engineered, synthetic biological processes. The domain insertion strategy used in this project could be considered a general approach by which researchers can tailor switches to their own requirements. The project will also provide a valuable insight concerning how apparently unrelated protein functions can be coupled, which will help us to understand how natural allosteric proteins evolved from simpler proteins and provide vital new information that will aid in the design of new allosteric proteins. This is currently being exploited by us and other groups
The project is also relevant to areas outside the biological sciences. The project aims to couple the chemistry of a small molecule to the function of a protein so will be of direct relevance to researchers in the area of chemical biology. As proteins work at the nanometre scale, their application as a molecular switch has direct relevance to nanotechnology. As this project will demonstrate that proteins with no direct relationship in nature can be coupled, it will open up the possibility of researchers identifying protein functions of significance to nanotechnology and linking their functions to produce a suitable device or material. This will allow researchers to create novel sensors, transducers and intelligent materials that respond in a required manner rapidly and reversibly with high temporal and spatial resolution.
Sectors Chemicals,Education,Pharmaceuticals and Medical Biotechnology,Other

URL http://www.cardiff.ac.uk/news/articles/protein-engineers-2498.html
 
Description The main impact of the findings are: (i) Development and demonstration of a general protein mutagenesis technology. The underlying whole gene random domain insertion approach was successfully implemented and extended to sample different mutational events during the course of the grant. (ii) to pursue commercialisation funding via the award of a Follow-on-Fund grant (BB/FOF/263) (iii) to provide exemplification for a patent application [Jones DD (Cardiff University). A directed evolution technology for altering the amino acid sequence of a protein. WO 2006077411] (iv) use of the data by the BBSRC as part of their 2008 "Roadshows" to exemplify the fledgling area of Synthetic Biology. (v) a demonstrator in public engagement events (e.g. science cafe) for the uses of synthetic biology (vi) as the basis for lectures to final year students as examples of "research-led teaching".
First Year Of Impact 2008
Sector Education
Impact Types Economic,Policy & public services

 
Description Defra Science note. SynBio
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description BBSRC-facing studentship
Amount £90,000 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 10/2017
 
Description Cardiff Partnership Fund
Amount £37,608 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 06/2008 
End 02/2009
 
Description Cardiff Synthetic Biology Initiative
Amount £45,108 (GBP)
Organisation SynbiCITE 
Sector Academic/University
Country United Kingdom
Start 05/2014 
End 07/2015
 
Description Constructing genetically encoded biosensors
Amount £94,446 (GBP)
Funding ID BB/FOF/263 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2010 
End 01/2011
 
Description Sparking with Impact Award
Amount £16,965 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2014 
End 06/2014
 
Title Transposon-directed evolution 
Description I have developed a series of protein mutagenesis technologies based on work at Cardiff that has been patented (WO 2006077411). The patent uses an engineered transposon to introduce random but defined breaks into a target gene that undergoes further downstream manipulations to generate mutations not sampled by existing directed evolution approaches. These include, trinucleotide deletion, trinucleotide replacement, TAG scanning mutagenesis and domain insertion. 
Type Of Material Biological samples 
Year Produced 2008 
Provided To Others? Yes  
Impact My research group has used the method to construct new and useful protein variants (see publication list) and has formed the basis for future grant funding. Other groups are also exploiting the method to generate useful protein variants. 
 
Description Merck KGaA BBSRC DTG Quota Case 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC)
Country United Kingdom 
Sector Public 
PI Contribution We introduced the general concept and the methodology by which to construct potentially useful protein sensors systems through the use of the domain insertion strategy. The project would be related to the construction of GFP-based sensing systems by which GFP fluorescence would be modulated by secondary events.
Collaborator Contribution They provided expertise in fluorescence and access to equipment at their German base in Darmstadt for fluorescence life-time measurement.
Impact The most useful output has been several papers and a trained PhD student (Arpino). Papers (DOIs): 10.1007/978-1-4939-1053-3_11 10.1039/b904031e 10.1021/ja301987h 10.1016/j.str.2014.03.014 10.1371/journal.pone.0047132 10.1107/S139900471401267X
Start Year 2007
 
Description Merck KGaA BBSRC DTG Quota Case 
Organisation Merck
Country Germany 
Sector Private 
PI Contribution We introduced the general concept and the methodology by which to construct potentially useful protein sensors systems through the use of the domain insertion strategy. The project would be related to the construction of GFP-based sensing systems by which GFP fluorescence would be modulated by secondary events.
Collaborator Contribution They provided expertise in fluorescence and access to equipment at their German base in Darmstadt for fluorescence life-time measurement.
Impact The most useful output has been several papers and a trained PhD student (Arpino). Papers (DOIs): 10.1007/978-1-4939-1053-3_11 10.1039/b904031e 10.1021/ja301987h 10.1016/j.str.2014.03.014 10.1371/journal.pone.0047132 10.1107/S139900471401267X
Start Year 2007
 
Description Single protein molecule ET and nanotech. EP/J015318/1 
Organisation Cardiff University
Country United Kingdom 
Sector Academic/University 
PI Contribution The generation of engineered proteins for use in protein-gold and protein-sp2 system interfacing.
Collaborator Contribution Single protein molecule measurement and analysis
Impact This is a multi-disciplinary project involving the School of Bioscience and Physics. Recent papers (DOI): 10.1039/c2cc34302a 10.1002/smll.201102416 10.1039/c2nr32131a 10.1021/nn2036818 10.1021/nl103334q
Start Year 2006
 
Description Single protein molecule ET and nanotech. EP/J015318/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC)
Country United Kingdom 
Sector Public 
PI Contribution The generation of engineered proteins for use in protein-gold and protein-sp2 system interfacing.
Collaborator Contribution Single protein molecule measurement and analysis
Impact This is a multi-disciplinary project involving the School of Bioscience and Physics. Recent papers (DOI): 10.1039/c2cc34302a 10.1002/smll.201102416 10.1039/c2nr32131a 10.1021/nn2036818 10.1021/nl103334q
Start Year 2006
 
Title POLYPEPTIDE MUTAGENESIS METHOD 
Description There is provided a method for altering the amino acid sequence of a target polypeptide by altering a target DNA sequence which encodes that polypeptide, the method comprising the step of introducing a transposon into the target DNA sequence, in which the transposon comprises a first restriction enzyme recognition sequence towards each of its termini, the recognition sequence not being present in the remainder of the transposon, or in the target DNA sequence, or in a construct comprising the target DNA sequence, the first restriction enzyme recognition sequence being recognised by a first restriction enzyme which is an outside cutter and being positioned such that the first restriction enzyme has a DNA cleavage site positioned beyond the end of the terminus of the transposon. 
IP Reference WO2006077411 
Protection Patent application published
Year Protection Granted 2006
Licensed Yes
Impact The generation of new and useful fluorescent proteins.
 
Description School Visit (Wales) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact talk and discussion with students

Informed students on current science
Year(s) Of Engagement Activity 2009
 
Description Talks at Universities. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Talks to students, PhDs, postdocs and PIs in areas related to those associated with the grants.

Collaborations.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013