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.

               

E. coli Infection

Reference

Zhang et al. “A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance.” Nature Chemical Biology (2011) 7, pgs 671 – 677

                For the Gram-negative bacterium Escherichia coli to successfully infect a victim following ingestion, it must survive a trip through the stomach and reach the small intestine.  Mammalian stomachs create a low pH environment to help break down incoming proteins from both food eaten and any accidently ingested pathogens.  Unfortunately, several enteric bacteria, including some strains of E. coli, are able to survive the acidic stomach to arrive at the neutral small intestine intact and successfully infect a victim.

                The outer membranes of Gram-negative bacteria are quite porous and will allow passage of molecules smaller than 600 Da.  Obviously protons can easily cross that membrane to reach the periplasmic proteins within.  How do the bacteria protect these proteins from either denaturation at low pH (stomach) or incorrect renaturation upon reaching neutral pH (small intestine)?

                It was previously known that the bacterial protein HdeA binds periplasmic bacterial proteins at low pH to protect them.  Once reaching the neutral small intestine, HdeA releases its substrates in a nonactive form that must then be properly folded again for full function.  What additional chaperones were involved in this process as well as substrates for HdeA were unknown.

                Recent work by Chen and colleagues, published last month in the journal Nature Chemical Biology, focused on identifying substrates for HdeA by using an unnatural amino acid (named DiZPK by the authors) whose side chain can photocrosslink with proximal protein.  They were able to place this version of HdeA inside living E. coli cells, subject them to low pH and thus identify substrates for HdeA.

                Interestingly, the two substrate proteins identified here are DegP and SurA, both of which are essential chaperone protein themselves.  The authors theorize that HdeA exists to protect these two important chaperones at low pH and helps refold them upon neutralization, which means they are then subsequently free to help other proteins refold (Figure 4.1, directly from their paper).  While the cytosol has mechanisms in place for chaperone protein folding mediated by ATP, the periplasmic space is low in ATP so the bacteria have developed another way to circumvent the situation.

                The importance of HdeA could lead to new therapies to treat E. coli infections.