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Department of Pharmacology




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.

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.

We use a battery of techniques spanning protein engineering, single-molecule methods, biophysics, biochemistry and analysis in cellulo and in silico.

In combination with site-directed mutagenesis, this approach allows us to probe a mechanism or process and find out whether there are overarching, fundamental laws that govern it, which should in turn help 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.

Our group and others have shown that the simple modular, 1D-like architecture of 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.

Perez-Riba, A., M. Synakewicz, and L. S. Itzhaki. Opinion piece: Folding cooperativity and allosteric function in the tandem-repeat protein class. Phil. Trans. R. Soc. B, 373:20170188, 2018.


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 exploring three different approaches to target them:

  • chemically constrained peptides
  • designed repeat proteins
  • 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 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.

Burbidge O., M. W. Pastok, S. L. Hodder, G. Zenkevičiūtė, M. E. M. Noble, J. A. Endicott, and L. S. Itzhaki. Nanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a.

Diamante, A., P. Chaturbedy, P. Rowling, J. Kumita, R. S. Eapen, S. H. McLaughlin, M. de la Roche, A. Perez-Riba, and L. S. Itzhaki. Engineering mono- and multi-valent inhibitors on a modular scaffold. Chem. Sci., 12(3):880-895, 2021.

Xu, W., Y. Heng Lau, G. Fischer, Y. S. Tan, A. Chattopadhyay, M. de la Roche, M. Hyvönen, C. S. Verma, D. R. Spring, and L. S. Itzhaki. Macrocyclized extended peptide Inhibiting the substrate-recognition domain of tankyrase. J. Am. Chem. Soc., 139(1):2245-2256, 2017.


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 be 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.

Synakewicz M., R. S. Eapen, A. Perez-Riba, D. Bauer, A. Weißl, G. Fischer, M. Hyvönen, M. Rief, L. S. Itzhaki, and J. Stigler. Consensus tetratricopeptide repeat proteins are complex superhelical nanosprings.


Repeat protein assembly and nano-materials

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 carry out new functions.

Ripka J. F., A. Perez-Riba, P. K. Chaturbedy, and L. S. Itzhaki. Testing the length limit of loop grafting in a helical repeat protein. Curr. Res. Struct. Biol., 3(1): 30-40, 2021.

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.