Knotting and Linking in Topologically Complex Proteins

A covalently and topologically linked dimer, citrate synthase, in the hyperthermophile P. aerophilum. (Adapted from Boutz, et al.)
A covalently and topologically linked dimer, citrate synthase, in the hyperthermophile P. aerophilum. (Adapted from Boutz, et al.)

Although proteins often adopt rather complex folds, the fact of the matter is that the backbone of a protein molecule adopts a knotted configuration in only a very few proteins among the thousands of known protein folds. This is contrary to what would be expected if protein backbones followed essentially random paths in space; for spatial curves as long as medium-sized proteins, knots would be expected to dominate. The observation that native protein folds hardly sample the space of knotted conformations – a space that represents the majority of all possible conformations – has profound implications for protein folding in general, and for the Thermodynamic Hypothesis (first posed by Anfinsen) in particular.

Discovery of the first protein slip knot, in the thermophilic enzyme alkaline phosphatase. The panel on the right shows a knot plot that revealed that a partial structure within the intact fold forms a knot. (Adapted from King, et al. (2007))
Discovery of the first protein slip knot, in the thermophilic enzyme alkaline phosphatase. The panel on the right shows a knot plot that revealed that a partial structure within the intact fold forms a knot. (Adapted from King, et al. (2007))

Despite their rarity, there are a few special cases where knotted proteins have been observed, and these provide special opportunities for studying protein folding and stability. The intuition that it might be kinetically difficult for a knotted protein chain to reach its native state can be considered in reverse, with the conclusion that knotting and other kinds of topological entaglements could kinetically stabilize the folded states of proteins containing these features. While looking for unusual topological features in protein stuctures, we (Neil King) discovered a novel slipknot feature in a well-known thermostable protein, alkaline phosphatase. Beyond the knotting of individual protein chains, a few rare cases are known in which protein chains from two different subunits form topologically interlinked chains. One of these was discovered in the lab by Danny Boutz in the citrate synthase dimer from P. aerophilum.

Design of a novel knotted protein fold. Protein folding experiments demonstrate that the knot substantially complicates the folding landscape and retards folding. (Adapted from King, et al. 2010).
Design of a novel knotted protein fold. Protein folding experiments demonstrate that the knot substantially complicates the folding landscape and retards folding. (Adapted from King, et al. 2010).

In recent work we have used protein design to generate novel types of protein topologies in order to test the consequences of topological complexity on protein folding, and as a potential avenue for creating interesting types of protein-based materials.

Polymerizing a shallowly knotted protein produces a series of deep knots, which stabilizes the protein compared to the control. (Adapted from Sayre, et al.)
Polymerizing a shallowly knotted protein produces a series of deep knots, which stabilizes the protein compared to the control. (Adapted from Sayre, et al.)

References:

2011

Sayre TC, Lee TM, King NP, Yeates TO
Protein stabilization in a highly knotted protein polymer.
Protein Eng. Des. Sel.. Aug 2011. 24(8):627-30. 2011 PMID: 21669955
PMC3165941 10.1093/protein/gzr024

2010

King NP, Jacobitz AW, Sawaya MR, Goldschmidt L, Yeates TO
Structure and folding of a designed knotted protein.
Proc. Natl. Acad. Sci. U.S.A.. Nov 2010. 107(48):20732-7. 2010 PMID: 21068371
PMC2996448 10.1073/pnas.1007602107

2007

Yeates TO, Norcross TS, King NP
Knotted and topologically complex proteins as models for studying folding and stability.
Curr Opin Chem Biol. Dec 2007. 11(6):595-603. 2007 PMID: 17967433
PMC2179896 10.1016/j.cbpa.2007.10.002 NIHMS35923

2006

Norcross TS, Yeates TO
A framework for describing topological frustration in models of protein folding.
J. Mol. Biol.. Sep 2006. 362(3):605-21. 2006 PMID: 16930616
10.1016/j.jmb.2006.07.054

structural, computational, and synthetic biology