Tying up the Loose Ends: A Mathematically Knotted Protein

Area of Science:

• Biophysics
• Polymer Physics
• Protein Science

Background:

• Protein knots are prevalent but their formation and function remain unclear.
• Existing protein knots differ from mathematical knots due to open termini.
• Characterizing polymer knotting is challenging due to difficulties in controlling unfolded states.

Purpose of the Study:

• To create and investigate a truly backbone-knotted protein.
• To directly probe the effects of backbone knots and reduced conformational entropy on protein folding.
• To elucidate the contributions of knot structure versus cyclization to protein stability and properties.

Main Methods:

• Enzymatic peptide ligation to covalently link termini of YibK protein, creating a backbone knot.
• Production and investigation of backbone-cyclized YibK without a knot for comparison.
• Analysis of thermal stability, cofactor binding, and aggregation propensity.

Main Results:

• Backbone cyclization did not alter native structure or cofactor binding.
• Thermal stability significantly increased, and aggregation propensity decreased in cyclized YibK.
• Enhanced stability is attributed to a more rugged free energy landscape and destabilization of the denatured state, with minimal contribution from the knot structure itself.

Conclusions:

• Backbone cyclization is a viable method to create truly knotted proteins.
• Protein knot structure has a minor role in enhanced stability; landscape changes are dominant.
• Results align with polymer physics predictions for chemically unfolded cyclized proteins.