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A tangled problem: how does a knotted protein fold?

Final Activity Report Summary - KNOTTEDPROTEINFOLD (A tangled problem: how does a knotted protein fold?)

The folding of proteins that have knots in their polypeptide chain is a challenging new problem within the protein folding field. The first knotted protein of this kind to have its folding pathway characterised was the methyltransferase YibK in the Jackson laboratory in Cambridge. To further investigate the effect of knots on protein folding pathways, circular permutants (CPs), proteins in which the original N- and C-termini of the amino acid sequence are linked together, and new N- and C-termini are created elsewhere in the sequence, of YibK were constructed. This strategy is of particular interest in the case of knotted proteins, as it allows the removal of the knot or the modification of the length of the polypeptide chain passing through the knot. A comparative study of the stability and folding of the novel CPs with wild-type protein would provide important information on how and when the knot formed during folding.

Several circular permutant constructs were designed and made using molecular biology techniques, however, most of these constructs were unable to fold correctly, and aggregated in inclusion bodies during their expression. Our study revealed that the addition of a linker between both original N- and C-termini and/or the creation of new ends can have a drastic effect on the stability and folding of proteins in general and knotted proteins in particular. The screening and set up of a series of linker with different lengths and amino acid composition, as well as alternative purification protocols could help to obtain soluble circular permutants constructs.

Although the premature end of the Fellowship, after one year of funding, did not allowed the completion of the planned work, a study of the folding of another knotted protein, the human Ubiquitin C-terminal hydrolase UCH-L3 was initiated and interesting and important preliminary results obtained. UCH-L3 has the particularity of being one of the few knotted proteins identified so far in humans which has a complex Gordian knot with two topological crossings (in comparison with the simple trefoil knot of YibK). Mutations in the homologue UCH-L1 are known to be associated with Parkinson's disease. Characterising the folding pathway of such structures is, therefore, crucial not only for our general understanding of how knotted proteins fold but also how mutations of such structures affects stability and folding, and results in diseased states.