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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