Tandem-repeat proteins: folding and function
A major aim of our current research is to define, using a battery of techniques spanning single molecule methods, biophysics, chemical biology and analysis in cellulo and in silico, the conformational transitions that 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. In order to achieve our goals a major focus of our efforts is the class of proteins known as tandem-repeat proteins. They comprise small structural units repeated multiple times in tandem to form non-globular, elongated structures that present extended scaffolds for molecular recognition (Fig. 1). The term “scaffold” implies a rigid architecture; however, as suggested by their Slinky spring-like shapes, it is proposed 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; proteins that wrap around their cargoes to transport them between the nucleus and cytoplasm. Our group and others have shown that the simple modular, one-dimensional-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 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 (Fig. 2).
We are focusing on ankyrin, HEAT and ARM 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 (e.g. Fig. 3). Ankyrin repeats have also been identified as targets in a range of diseases in addition to cancer. For example, antagonists of ankyrin repeat ion channels have shown potential in pain relief and in treating respiratory diseases, chronic acid reflux and esophageal hypersensitivity.
The properties of the individual units of tandem 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.) and also with multi-functionality; such a degree of rational engineering is not possible with globular proteins. We are interested in exploiting this extraordinary design-ability to create a toolkit of parts with which to build novel proteins for used in medicine and biotechnology.
Molecular pathology of cancer-associated missense mutations?
Previous in vitro analysis suggests that most of the cancer-causing missense mutations in tumour suppressors p16 (an ankyrin repeat protein) and BRCA1drastically destabilise the structures (Fig. 4), thereby causing loss of function. Our aim is now to understand how these proteins respond to mutations when they are surrounded by the complex environment of the cell.