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Research

 

repeat

A major focus of our research is a class of proteins known as tandem-repeat proteins. They comprise small structural units repeated multiple times in tandem to form non-globular, elongated and superhelical structures that present extended scaffolds for molecular recognition. There is a large variety of repeat types ranging from purely helical or beta-sheet to a mix of both. Consequently, they give rise to many different geometrical shapes, which in turn confer different biochemical and biophysical properties and functions. thereby presenting an extended scaffold for molecular recognition. The term ‘scaffold’ implies a rigid architecture; however, as suggested by their spring-like shapes, it is thought that repeat arrays utilise much more dynamic and elastic modes of action. For example: stretching and contraction motions to regulate the activity of a bound enzyme; reversible nanosprings to operate ion channels; transporters that wrap around their cargoes to carry them in and out of the nucleus.

repeat types
Different types of repeats (from left to right): HEAT, TALE, Ankyrin, TPR, Armadillo, β-helix
In our lab, we use a battery of techniques spanning protein engineering, single-molecule methods, biophysics, biochemistry and analysis in cellulo and in silico. These techniques, in combination with site-directed mutagenesis allow us to probe a mechanism or process and find out whether there are overarching, fundamental laws that govern it. As such they provide a bottom-up approach: learning more about the molecular characteristics of a given system will enable us to guide drug design and therapeutic intervention. At the same time, we are also investigating novel approaches to disease treatment based on, and motivated by, what we know about the underlying principles of repeat-protein structure and function.

 

Structure, function and mechanics of repeat proteins

One major aim of our current research is to define the conformational transitions that repeat proteins undergo during the different stages of their life cycle - biosynthesis, folding, localisation, assembly and degradation - and how they are guided by the cellular machinery. Due to their modular architecture and spring-like shapes, it is proposed that repeat arrays utilise much more dynamic and elastic modes of action than globular proteins. For example: stretching and contraction motions to regulate the activity of a bound enzyme; acting as reversible nanosprings to operate ion channels; proteins that wrap around their cargoes to transport them between the nucleus and cytoplasm.

Our group and others have shown that the simple modular, 1D-like architecture of tandem repeat proteins gives them distinctive properties compared with globular proteins and makes it uniquely straightforward to map the energetics of their structures and also to rationally redesign their stability, folding and molecular recognition. This class of proteins is thus an exceptionally sensitive and versatile tool that we are now in a position to exploit to dissect otherwise intractable cellular mechanisms.

 

crystal
TNKS2 ARC4 bound to stapled peptides

Protein design and molecular therapeutics

We are focusing on repeat proteins that play important roles in disease, in particular cancer, and we are using the insights we obtain to develop therapeutic strategies for targeting them. In our group, we are exploring three different approaches to target our proteins of interest: (i) chemically constrained peptides,
(ii) designed repeat proteins, and (iii) nanobodies (single-domain antibodies).

In most cases, the proteins that we are targeting are repeat proteins or repeat-protein domains that are central to cellular function, organism development and homoeostasis. Targets include the substrate-binding subunits of oncogenic E3 ubiquitin ligases and the substrate-recognition domain of the poly(ADP) ribose polymerase tankyrase. In other projects, we are engineering new functions into ultra-stable consensus-designed repeat proteins with the aim to disrupt aberrant protein-protein interactions or to remove proteins upregulated in disease. Lastly, cancer-associated mutations can result in loss of function by reduced protein stability; the tumour suppressor p16INK4a is such an example, and we are investigating the use of nanobodies to stabilize these disease-associated p16 variants and thereby restore function.

 

Designed repeat proteins as tension sensors for mechanotransduction research

Forces are key to a wide variety of cellular processes, and It is clear that specific proteins must be able to sense mechanical signals and convert them into biological responses. However, a molecular-level understanding of such processes is very limited, as accurate measurement of the forces poses significant challenges, in particular because it is currently not possible for us to build force sensor with tailored mechanical characteristics. We believe that repeat proteins should be able to address this challenge. Our group and others have shown that these proteins behave like molecular “nanosprings” and, because of the modular structure, they can put together like Lego from small structural motifs into large arrays. Using these nanosprings, we intend to produce a toolkit of force sensors that can be customised for diverse in cellulo applications.

 

Repeat protein assembly and nano-materials

main2018
Assembly of protein cages using TPRs and split intein-mediated native chemical ligation

Repeat proteins have been observed to associate with binding partners having a variety of structures: some bind intrinsically disordered proteins or short linear motifs, some bind globular structures or other repeat proteins. The properties of the individual units of repeat proteins can be tailored by design and they can then be combined in a modular fashion to create artificial proteins with predictable properties (stability, binding etc.). Such a degree of rational engineering is not possible with globular proteins. We are interested in exploiting this extraordinary design-ability and functional variety with the ultimate goal of developing scaffolds that can bind or encapsulate specific peptides or small molecules. We also envisage scaffolds that could perform a single function or simple biological process in itself. Different approaches are used in our group to create these scaffolds and nano-materials; they include CuAAC (“click”) chemistry, protein-protein interactions and metal binding systems.

We have a range of investigations ongoing. For example, we have found a novel way of functionalizing amyloid fibrils using “click” chemistry. Fibrils with alkyne moieties displayed on their surface have been covalently linked to fluorophores. We are now working on linking different biomolecules, such as designed repeat proteins, to the fibrils such that the resulting nanomaterials can have a specific function.

reshaping
Four amino acid substitutions change the superhelical geometry

 

In another project, we are focusing on tetratricopeptide repeats (TPRs). The current limitation of natural TPRs to applications as scaffolds in biotechnology lies with their intrinsic properties such as stability and solubility. We are working on altering the shape of consensus-designed TPRs (CTPRs) to understand how sequence translates into geometry and to function and ultimately how we can rationally manipulate the scaffold properties by altering the shape of the constituent building blocks.