Single alpha helical domains: designing artificial levers for biological molecules

DNA encodes the sequences of proteins, which consist of chains of amino acids that fold up into a precise 3 dimensional structure. Proteins are essential for life, and the structures of different proteins specify how they work in cells, for example as an enzyme or as a structural protein. Discovering the principles that govern how proteins fold up into their three dimensional structure, is therefore key to understanding life, in understanding what goes wrong in disease, and in designing new proteins with useful properties that would, for instance, enable us to target man-made drugs to disease targets such as cancerous cells. Folded proteins contain a series of well-defined structural building blocks. One of these is the alpha helix, which looks a little like a coiled spring. To understand how proteins fold up into their three dimensional structure, the properties of alpha-helices have been intensively studied. In most cases, alpha-helices are not very stable by themselves in water: they collapse into a randomly folded chain. However, we have recently found that there is one type of naturally occurring alpha helix that is remarkably stable all on its own in water. This stable alpha helix is found in a wide variety of proteins from bacteria to humans, including in proteins important for cell movement and cell division. At the moment, we know very little about what makes it so stable, or what its function in protein is. It is usually found sandwiched between two regions of the protein in such a way that we think they might act as stiff linkers (or struts) between the two parts of the protein, and transmit information about changes in structure in one part of the protein to the other part. We want find out what makes these alpha helices so stable, and to test our ideas about what they do, by determining the mechanical properties of artificial and naturally occurring alpha-helices that we think form stable alpha helices in water. To learn as much as possible in a short time, we want to have two researchers with different skills working together to study these helices one molecule at a time. One of them will engineer a series of proteins containing these alpha helices and determine how their function depends on the sequence of the amino acids. The other researcher will find out the mechanical properties of each of these engineered proteins using a new apparatus that lets us study how a single molecule unfolds when it is stretched and refolds when it is released, and will also create models in the computer to explain the results and predict new properties. They will also send these proteins to our collaborators in the USA who can discover additional properties of them using their own specialized methods. By using as wide a range of techniques as possible, to study this stable alpha helix, we think we will determine why it is so stable, and what proteins need it for.

Single alpha-helical (SAH) domains are single alpha helices, which are rich in both acidic and basic amino acid residues, and are stable in solution in isolation. We have shown that sequence analysis software often mis-identifies them as forming coiled coils, but they lack the hydrophobic seam required for this behaviour. They are widespread in a range of proteins in the human genome, and widespread in Nature, occurring in organisms from bacteria to humans, where they are typically found sandwiched between two functional domains in these proteins. The significance, and functional role of the SAH domains are only just starting to be appreciated, and we have much to learn about their mechanical stability and how this contributes to their function in proteins. We recently showed that SAH domains are stiff enough to act as a lever in the molecular motor myosin, by amplifying small changes in the motor domain into a large movement of cargo. We suspect that this property of SAH domains is a general property and may be used in other proteins to convert small conformational changes into large movements between domains via movement of this structural element. The stability of the SAH domain in water also allows us to test the mechanical properties of an alpha-helix in isolation for the first time. The aim of our research is to characterize the mechanical properties of a range of artificially designed and naturally-occurring SAH domains using a wide range of single molecule biophysical techniques to characterise this little-known structural motif and determine what contributes to its stability, and how its properties are important for function in proteins. An additional outcome will be the potential to use SAH domains in the rational design of proteins for novel applications.

The academic community will be the main beneficiaries from this research, mainly through the new knowledge that we will obtain on the structure and function of this novel stable single alpha-helical (SAH) domain. As this domain is present in a wide range of proteins, the research is likely to be of broad general interest. The two main ways that we will ensure that they have the opportunity to benefit from this research, will be by presenting data at large international meetings, and through scientific publications, both research papers and reviews, in high impact journals that publish scientific findings of broad relevance. As our knowledge of the single alpha helical domain is at a very early stage, the research is not likely to have an immediate impact outside of the academic community. However, the research findings are likely to have longer term direct and indirect impacts as they will lead to a better understanding of how proteins fold in general, and the properties of the single stable alpha helix in particular, which has the potential to be developed for bio-nanotechnological applications. The University of Leeds, and the Faculties of Biological Science (FBS), and of Maths and Physical Sciences (MAPS), all have staff dedicated to helping researchers pursue any potential health and economic impacts of their research, an example being the successful spin out company headed up by Prof. Ingham in FBS and John Fisher in Engineering, called Regenix. The University Enterprise and Innovation office supports staff in knowledge transfer efforts, and we will use their expertise to determine potential ways forward. The Staff and Departmental Developmental Unit (SDDU) and the individual faculties both run workshops and courses aimed at postdoctoral researchers, academics and postgraduate students to develop their enterprise awareness and promote an understanding of intellectual property. Everyone in involved with the proposed project will be encouraged to engage with this workshops. The Faculties also run Enterprise and Innovation events such as open days in which companies are invited to visit and learn about the research that is being carried out in the faculty. We plan to present a poster/display at these open days to capitalise on potential link ups with industry. The University Press office, together with Campus PR are very effective in communicating key research findings with high impact. We have already worked with them to generate press releases communicating our research, which was subsequently reported in the media. Finally, if successful, this research will train 2 postdoctoral researchers in a range of cutting edge and interdisciplinary research techniques that will enable them to contribute more effectively to the wider economy in the future.