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.
