1. Flexibility of GroES mobile loop is required for efficient chaperonin function
Tatsuya Nojima, Takahisa Ikegami, Hideki Taguchi, Masasuke Yoshida J Mol Biol. 2012 Sep 14;422(2):291-9. doi: 10.1016/j.jmb.2012.05.026. Epub 2012 May 25.
Chaperonin GroEL and its partner GroES assist the folding of nascent and stress-damaged proteins in an ATP-dependent manner. Free GroES has a flexible "mobile loop" and binds to GroEL through the residues at the tip of the loop, capping the central cavity of GroEL to provide the substrate polypeptide a cage for secure in-cage folding. Here, we show that restriction of the flexibility of the loop by a disulfide cross-linking between cysteines within the loop results in the inefficient formation of a stable GroEL-polypeptide-GroES ternary complex and inefficient folding. Then, we generated substrate proteins with enhanced binding affinity to GroEL by fusion of one or two SBP (strongly binding peptide for GroEL) sequences and examined the effect of disulfide cross-linking on the assisted folding. The results indicate that the higher the binding affinity of the substrate polypeptide to GroEL, the greater the contribution of the mobile loop flexibility to efficient in-cage folding. It is likely that the flexibility helps GroES capture GroEL's binding sites that are already occupied by the substrate polypeptide with various binding modes.
2. The disordered mobile loop of GroES folds into a defined beta-hairpin upon binding GroEL
F Shewmaker, K Maskos, C Simmerling, S J Landry J Biol Chem. 2001 Aug 17;276(33):31257-64. doi: 10.1074/jbc.M102765200. Epub 2001 Jun 6.
The GroES mobile loop is a stretch of approximately 16 amino acids that exhibits a high degree of flexible disorder in the free protein. This loop is responsible for the interaction between GroES and GroEL, and it undergoes a folding transition upon binding to GroEL. Results derived from a combination of transferred nuclear Overhauser effect NMR experiments and molecular dynamics simulations indicate that the mobile loop adopts a beta-hairpin structure with a Type I, G1 Bulge turn. This structure is distinct from the conformation of the loop in the co-crystal of GroES with GroEL-ADP but identical to the conformation of the bacteriophage-panned "strongly binding peptide" in the co-crystal with GroEL. Analysis of sequence conservation suggests that sequences of the mobile loop and strongly binding peptide were selected for the ability to adopt this hairpin conformation.
3. Improved soluble expression of the gene encoding amylolytic enzyme Amo45 by fusion with the mobile-loop-region of co-chaperonin GroES in Escherichia coli
Lei Wang, Hildegard Watzlawick, Olafur Fridjonsson, Gudmundur Hreggvidsson, Josef Altenbuchner Biocatal Biotransformation. 2013 Nov;31(6):335-342. doi: 10.3109/10242422.2013.858712. Epub 2013 Nov 25.
The gene encoding the amylolytic enzyme Amo45, originating from a metagenomic project, was retrieved by a consensus primer-based approach for glycoside hydrolase (GH) family 57 enzymes. Family 57 contains mainly uncharacterized proteins similar to archaeal thermoactive amylopullulanases. For characterization of these family members soluble, active enzymes have to be produced in sufficient amounts. Heterologous expression of amo45 in E.coli resulted in low yields of protein, most of which was found in inclusion bodies. To improve protein production and to increase the amount of soluble protein, two different modifications of the gene were applied. The first was fusion to an N-terminal His-tag sequence which increased the yield of protein, but still resulted in high amounts of inclusion bodies. Co-expression with chaperones enhanced the amount of soluble protein 4-fold. An alternative modification was the attachment of a peptide consisting of the amino acid sequence of the mobile-loop of the co-chaperonin GroES of E.coli. This sequence improved the soluble protein production 5-fold compared to His6-Amo45 and additional expression of chaperones was unnecessary.
