Protein knotting through concatenation significantly reduces folding stability

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Engineered knotted proteins represent a novel frontier in protein design.
• The HP0242 homodimer from Helicobacter pylori serves as a model for creating such structures.
• Understanding the impact of engineered knots on protein stability and folding is crucial.

Purpose of the Study:

• To investigate the structural and kinetic consequences of covalently linking two protomers of the HP0242 homodimer to create an engineered knotted protein.
• To assess the thermodynamic stability and folding pathways of the concatenated protein compared to its parent homodimer.

Main Methods:

• X-ray crystallography was used to analyze the native and concatenated protein structures.
• Nuclear Magnetic Resonance (NMR) hydrogen-deuterium exchange analysis was employed to probe structural stability in solution.
• Chemical shift perturbation mapping was utilized to identify regions affected by concatenation.

Main Results:

• Concatenation resulted in the first engineered knotted protein, with no apparent structural changes observed via X-ray crystallography.
• Folding kinetics were significantly slower for the concatenated knotted protein compared to the parent homodimer.
• NMR analysis revealed significant destabilization of the knotted structure in solution, with some regions destabilized by up to 5 kcal/mol.
• Chemical shift perturbations indicated that concatenation affects the protein in a manner similar to chaotropic agents.

Conclusions:

• The design strategy of creating protein knots by concatenation may be thermodynamically unfavorable.
• Covalent constraints imposed on flexible ends can lead to a rugged free energy landscape.
• This can increase the propensity for forming off-pathway folding intermediates, hindering efficient folding.