A molecular understanding of how stable single alpha helical domains behave as constant force springs in proteins.

Lead Research Organisation: University of Leeds
Department Name: Sch of Molecular & Cellular Biology


Proteins in cells are commonly exposed to forces. We have recently discovered a specialised protein domain that helps to separate different domains in a single protein, and we now think that it also protects them from unfolding when they are exposed to forces within the cell. This novel domain is a special type of alpha helix, one of the two main types of protein fold. Unlike other alpha helices, this type is highly stable by itself and its bending stiffness is reasonably high, which means it can act as a 'spacer'; when sandwiched between two domains in a protein, rather like a fairly flexible rod between two balls, in a weightlifting barbell. It separates those domains by a defined amount that simply depends on its length, as each residue is separated by 0.15nm in an alpha helix, and thus, the higher the number of residues, the longer spacer. We call this type of helix a 'SAH' domain (for stable single alpha helix).
Our latest data on this SAH domain suggest that while its bending stiffness is reasonably high, if it is pulled on (experiences forces) along its length, then it progressively unravels, and as it does so, it maintains force at a constant level. This is rather different to a spring, where the force would continue to rise as the ends of the spring are pulled apart. If the SAH domain acted as a spring, there would be a danger that the adjacent protein domains would also unfold if the force rose to a high enough level, damaging their function. In contrast, by maintaining a constant force, while the SAH is unfolding and elongating, the adjacent protein domains are protected from unfolding, and from detaching from the protein complex that they interact with, when they are exposed to forces.
What we now plan to do is to determine exactly what level of forces the SAH domain unfolds under, if it is exposed to this level of forces in a cell, and if so, if those forces really do unfold the SAH domain and make it unfold, as we suspect. We also want to determine the structure of the SAH domain, to understand how the charged amino acids in a typical SAH domain sequence interact, so we can understand at a molecular level, how the amino acids interact and enable this special type of alpha helix to be so stable.
This new work will give us unprecedented insight into the structure and function of the SAH domain, enabling us to exploit its properties in artificial proteins in the future.

Technical Summary

Stable single alpha-helical domains (SAH domains), specialized forms of alpha-helices rich in charged (E,K,R) residues, are widespread in the proteome, where they are sandwiched between functional domains in proteins. They play important functional roles in proteins. For example, we have discovered that they contribute to the mechanical lever of myosin motor proteins, able to replace the canonical 'IQ' based lever. This suggested that they could act as relatively rigid spacers, separating two functional domains in other SAH containing proteins. In new experiments, we have now tested their mechanical properties by single molecule force spectroscopy experiments and modelling, and this has shown that when SAH domains are extended, they act as constant-force springs, in that they are able to maintain tension while lengthening. This novel property is likely to be important for their functional role. It suggests that the SAH domain may unfold in vivo at low forces, and by doing so, protect adjacent functional domains from unfolding, and then refold when force is reduced. We now wish to capitalize on this finding and our lead in this area by using a multidisciplinary approach to address the following major aims: (a) determine the level of forces under which SAH domains unfold so that their roles in proteins can be understood and predicted, and to allow rational use of SAH domains in protein engineering; (b) determine the level of force that myosins are exposed to in the cell, and determine if the SAH domain unfolds under those forces (c) determine the structure and flexibility of SAH domains to understand their properties at the molecular level.

Planned Impact

The main beneficiaries of this research will be the academic community. The findings on the properties of SAH domains will be of interest to others working on proteins that contain SAH domains, from molecular motors to nuclear and Golgi proteins. It will also be of interest to synthetic biologists, as engineering in SAH domains into artificial proteins could be exploited to introduce stiff spacers with a defined length and force response between domains, which would separate domains, protect them from unfolding, but also enable refolding. Understanding the molecular basis of the stability of these unusual domains would enable 'better' SAH domains to be designed for use in this type of approach. The timescales for these types of benefit are likely to be in the short and in the longer term (1-20 years), as synthetic biology matures and develops.
The researcher on this project will learn a raft of new techniques and approaches, including NMR, crystallisation and structure determination, optical tweezers, and cellular imaging techniques such as FRET, developing their skill set, and enabling them to become an independent researcher in their own right. They will also be able to present their work at conferences, and as research papers and articles including reviews, how to manage their research, budget resources, and there is the potential for them to investigate collaborations with synthetic biologists as the research develops.


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Batchelor M (2015) Myosin tails and single a-helical domains. in Biochemical Society transactions

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Batchelor M (2019) Dynamic ion pair behavior stabilizes single a-helices in proteins in Journal of Biological Chemistry

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Batchelor M (2015) Effect of external pulling forces on the length distribution of peptides. in Biochimica et biophysica acta

Description We have investigated how a special type of structure behaves and is stabilised. All proteins are made up of two main types of structure, a beta sheet and/or an alpha helix. Most alpha helices are stabilised though a 3 dimensional fold within the protein. We work on a special type of alpha helix that is completely stable as a single alpha helix. These types of helices are stabilised through the particular types of amino acids that they contain. We've carried out new research that helps us to understand how these particular amino acids stabilise these novel structures, which are found in many types of proteins.
Exploitation Route Our findings help others understand the stability of similar structures that are found in many other proteins, and in the design of novel types of this domain.
Sectors Pharmaceuticals and Medical Biotechnology

Description Café-Scientifique debate 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was a debate funded by the BSA, on 'does it matter who funds science'. I was on the panel, and took part in a debate with the audience on this topic. There was a lot of discussion on who should fund science, if we can trust the outcomes of science, does it matter if it is industry or universities that do the science and so on. The conclusion was, that the audience did go away with a generally favourable view of how science is done, and that it is important for the public to fund it.
Year(s) Of Engagement Activity 2017
URL https://www.ypsyork.org/events/cafe-scientifique-debate-november/
Description Royal Society MP Pairing Scheme 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact I took part in the Royal Society MP Pairing Scheme in November 2015. I visited parliament, and shadowed an MP (Chris Green from Bolton West). Chris is due to make a return visit to the University in April 2016.
Year(s) Of Engagement Activity 2015,2016
URL https://royalsociety.org/grants-schemes-awards/pairing-scheme/