Protein Knotting

REFERENCE: Mallam and Jackson. “Knot formation in newly translated proteins is spontaneous and accelerated by chaperonins.” Nature Chemical Biology (2012) 8, pgs 147 – 153.

                YibK and YbeA are Escherichia coli proteins that are known to form trefoil knots (Figure 15.1) before folding to their full native structures.  Previous work has shown that these proteins will denature without losing their trefoil knot and spontaneously refold to a functional protein once placed in renaturing conditions.  Mallam and Jackson address in a recent paper published in Nature Chemical Biology how this trefoil knot is initially formed within the cell.

                The authors first worked in a cell free system using a coupled transcription/translation set up that only included the necessary components (tRNAs, rNTPs, enzymes, etc) and determined that in vitro translated protein behaved the same as bacterially expressed protein.  Using a pulse-proteolysis experimental set up, a time course of folding for the protein was determined.  Interestingly, a 10 – 20 minute lag time was seen between the translation and final protein folding and rate constants of 0.05 – 0.09 min^-1 were elucidated.  

                Curious if bacterial chaperones could help folding, GroEL in complex with GroES was added.  Strikingly, no delay was noted from translation to folded protein and rate constants increased by almost 20-fold.  Adding the chaperonin complex to denatured YibK and YbeA did not substantially increase the rate of folding leading to the conclusion that GroEL-GroES specifically facilitate trefoil knot formation.  An excellent overview of their work is shown in Figure 15.2, which is directly from the authors’ paper.

                Even under highly denaturing conditions, the undoing of the trefoil knot has never been observed.  It seems that proteins knot only once in their lifetimes.  This work gives scientists a step towards understanding protein folding.