Significance of Knotted Structures for Function of Proteins and Nucleic Acids - September 17-21, 2014

Significance of Knotted Structures for Function of Proteins and Nucleic Acids

Poster Session II

52 – POS Board 24 Characterisation of the Folding Pathway of a Topologically Knotted Human Ubiquitin Hydrolase Hongyu Zhang 1 , Alexander Reeve 1 , Shihchi Luo 2 , Shang-Te Hsu 2 , Sophie E. Jackson 1 . 1 University of Cambridge, Cambridge, United Kingdom, 2 Academia Sinica, Taipei, Taiwan. Proteins containing topological knots have been steadily increasing in numbers. Due to their complex topology, their folding mechanisms are less well characterised compared to small single-domain proteins. Earlier biophysical studies on proteins with simple trefoil knot have shown that they can fold efficiently via obligatory intermediates states. However, the folding of more complex knotted systems such as proteins with 41, 52 or 61 knots remains to be characterised. Human ubiquitin C-terminal hydrolase homolog L1 (UCH-L1), has one of the most complex knot topologies identified so far – a 52 so-called Gordian knot. Earlier equilibrium unfolding experiments have shown that an intermediate state is stably populated at moderate urea concentrations. However, characterisation of the folding pathway by kinetic studies has been impeded because the transition between the intermediate (I) and denatured (D) states is fast and the fluorescent signal change is very small. Therefore, we constructed a series of mutants, L3W, L70W, F108W, V113W, F160W, F181W, F214W, each of which contains a single tryptophan at different locations on the polypeptide chain which act as fluorescence reporter of local structure formation ( the tryptophan 26 in wild-type was substituted by a phenylalanine in the mutants). The equilibrium unfolding studies have shown that the mutants have the same m-values for each structural transition as wild type, suggesting that the substitutions do not significantly change the nature of the intermediate state. However, the stability of the mutants varies. Mutants F160W, F181W and F214W show enhanced fluorescence for the I-D transition and thus can be used to characterise the folding mechanism in further detail. The results of single jump and double jump unfolding and refolding measurements suggest that UCH-L1 folds via a parallel pathway on each of which an intermediate is populated.

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