The following post was written by our resident (self confessed) protein geek, and good friend, Laura Mitchell. Laura is in her final year of Biochemistry at UCL and also did her placement at MedImmune in the Analytical Biochemistry team working on the development of mass spectrometry methods for observing antibody dynamics (fancy stuff).
On first glance tandem repeat proteins (TRPs) may be easily dismissed as dull, or even interpreted as one of nature’s mistakes – perhaps caused by slippage in replication. They comprise tandem arrays of small structural motifs (20 – 40 amino acids), that pack linearly to form long, one-dimensional structures. A bit like the guys on this bike:
Except the domains can be repeated many times like this:
They occur throughout nature featuring in all kinds of proteins, mainly performing roles in binding other proteins and nucleic acids. As the old biochemical mantra goes; protein structure determines function – so what is interesting about such a repetitive structure in a simple topology? (other than looking pretty)…
Image taken from Dr Laura Itzhaki’s research page: http://www.ch.cam.ac.uk/person/lsi10
Unlike globular proteins such as GFP or haemoglobin, TRPs have very few stabilising interactions between residues that are far apart in sequence. This makes them relatively straightforward to manipulate. Protein analysis shows the vast majority of residues are involved in stabilising the architecture of the folds, with only a few select residues identified in other functions such as binding. This makes them even more straightforward to manipulate. Additionally, their regularity and modularity means the number (and type) of repeats can be modified for altered properties. Altogether, these attributes make them amenable to rational protein engineering – which is a big deal in the young field of protein engineering.
TRP’s display attributes of curvature and twist, which vary depending on the repeat module. For example, Leucine Rich Repeat proteins (LRRs) have large curvature and small twist, so form concave surfaces that can wrap around other protein domains. Armadillo repeat proteins have large twist and small curvature, and have grooves that can bind peptides.
An atomic force microscopy cantiliever (AFM) study (basically molecular-sized tweezers that can pick at a single immobilised protein molecule) by the Marszalek lab at Duke University has shown that Ankyrin repeat proteins are highly elastic, and that they can be stretched and relaxed repeatedly without showing any signs of wear. Hence, they have the mechanical properties of a nanospring! They could be used as nanoscale “shock absorbers” or for any application requiring elasticity on the nanoscale.
Credit: Piotr Marszalek, Duke University http://www.nature.com/nature/journal/v440/n7081/full/nature04437.html
Proteins are already routinely modified to improve properties such as binding, solubility or stability, but it is still an enormous challenge to engineer proteins to fold and function as we’d like them to. An important aim of protein engineering is to modify, create or inhibit protein-protein interactions, (which are involved in diagnostics, therapeutics and imaging). Traditionally antibodies have been used for many of these roles, but TRPs are a promising alternative with perhaps greater “designability”. Their potential to bind large surfaces and interrupt binding could also be an additional therapeutic mechanism of action to that achievable by antibodies. TRPs are also easily expressed at high yields in E. coli and tend to be very stable – not only any protein-enthusiast’s dream, but also key properties for application in biotechnology.
Biomaterial design is another field these proteins could be used in. An example taken from an excellent review by Ewan Main, Jonathan Phillips and Charlotte Millership, is the tetratricopeptide repeat (TPR) motif, which has been engineered (in simple but ingenious ways) to form “various levels of complexity from one-dimensional filaments to two-dimensional films and three-dimensional hydrogels”. In each case the core repeating structure has been manipulated to be expressed as monomers, which can self-assemble into constructions of controlled size and design, but only under trigger-conditions (such as the addition of a chemical cross-linker).
A) CTPR18 – Comprised of alternating peptide-binding and spacer repeat TPR domains B) Combination of CTPR18 with peptide-PEG crosslinker gives a 3D-hydrogel C) CTPR18 forms a thick film after being left to dry on a teflon surface with a plasticizer D) Chemically modified monomers can covalently bond in head-to-tail fashion to form oligomers, as shown in the transmission EM micrograph. Credit: E. Main, J. Phillips and C. Millership
Additional functionality can be engineered into these self-assembling constructs through the inclusion of useful moieties such as PEG. It could be envisaged that these modular proteins may be one day put together like lego – but a complex kind of lego with adaptable, responsive elements and functional extras. Personally I am excited by the possibilities of “plug and play” protein technology that could be available in the future.