title: Probing nature's knots: the folding pathway of a knotted homodimeric protein.
abstract: The homodimeric protein YibK from Haemophilus influenzae belongs to a recently discovered superfamily of knotted proteins that has brought about a new protein-folding conundrum. Members of the alpha/beta-knot clan form deep trefoil knots in their native backbone structure, a topological feature that is currently unexplained in the protein-folding field. To help solve the puzzle of how a polypeptide chain can efficiently knot itself, the folding kinetics of YibK have been studied extensively and the results are reported here. Folding was monitored using probes for changes in both secondary and tertiary structure, and the monomer-dimer equilibrium was perturbed with a variety of solution conditions to allow characterisation of otherwise inaccessible states. Multiphasic kinetics were observed in the unfolding and refolding reactions of YibK, and under conditions where the dimer is favoured, dissociation and association were rate-limiting, respectively. A folding model consistent with all kinetic data is proposed: YibK appears to fold via two parallel pathways, partitioned by proline isomerisation events, to two distinct monomeric intermediates. These form a common third intermediate that is able to fold to native dimer. Kinetic simulations suggest that all intermediates are on-pathway. These results provide the valuable groundwork required to further understand how Nature codes for knot formation.
authors.affiliation: Chemistry Department, Cambridge, UK.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Mallam
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Jackson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005453:Fluorescence; D006193:Haemophilus influenzae / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins