Abstract
Concatenation by covalent linkage of two protomers of an intertwined all-helical HP0242 homodimer from Helicobacter pylori results in the first example of an engineered knotted protein. While concatenation does not affect the native structure according to X-ray crystallography, the folding kinetics is substantially slower compared to the parent homodimer. Using NMR hydrogen-deuterium exchange analysis, we showed here that concatenation destabilises significantly the knotted structure in solution, with some regions close to the covalent linkage being destabilised by as much as 5 kcal mol−1. Structural mapping of chemical shift perturbations induced by concatenation revealed a pattern that is similar to the effect induced by concentrated chaotrophic agent. Our results suggested that the design strategy of protein knotting by concatenation may be thermodynamically unfavourable due to covalent constrains imposed on the flexible fraying ends of the template structure, leading to rugged free energy landscape with increased propensity to form off-pathway folding intermediates.
Highlights
The native structure of HP0242 to the extent that it resembles the global unfolding effect induced by concentrated chaotrophic agent, suggesting that knotting by concatenation may be thermodynamically unfavourable
We have previously reported the backbone NMR assignments of wt HP0242 and the secondary structures in solution state are consistent with the crystal structure28. {1H}-15N heteronuclear nuclear Overhauser effect of HP0242 confirmed that all the four helices are highly ordered on the timescale of ps-ns (Figure S1)
While the hydrogen-deuterium exchange (HDX) rates of most of the residues were proportional to catalyst concentration, i.e., [OH−], indicating that the HDX process is under thermodynamic equilibrium, known as the EX2 regime[31], a few residues showed pH independent HDX rates, indicating that the HDX of these residues are under kinetic control of the opening rates of the corresponding hydrogen bonds, known as the EX1 regime
Summary
The native structure of HP0242 to the extent that it resembles the global unfolding effect induced by concentrated chaotrophic agent, suggesting that knotting by concatenation may be thermodynamically unfavourable. For the EX2 residues, we calculated the protection factor (PF), and derived the corresponding free energy of unfolding, ΔGHDX, for individual backbone amide-mediated hydrogen bonds (see Methods).
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