Identification of proline residues responsible for the slow folding kinetics in pectate lyase C by mutagenesis

Biochemistry
Douglas E Kamen, Robert W Woody

Abstract

The folding mechanism of pectate lyase C (pelC) involves two slow phases that have been attributed to proline isomerization. To have a more detailed and complete understanding of the folding mechanism, experiments have been carried out to identify the prolyl-peptide bonds responsible for the slow kinetics. Site-directed mutagenesis has been used to mutate each of the prolines in pelC to alanine or valine. It has been determined that isomerization of the Leu219-Pro220 peptide bond is responsible for the slowest folding phase observed. The mutant P220A shows kinetic behavior that is identical to the wild-type protein except that the 46-s phase is eliminated. The Leu219-Pro220 peptide bond is cis in the native enzyme. An analysis of the free energy of unfolding of this mutant indicates that the mutation destabilizes the protein by about 4 kcal/mol. However, it appears that the major refolding pathways are unaltered. Further mutations were carried out in order to assign the peptide bond responsible for the 21-s folding phase in pelC. Mutation of the remaining 11 prolines, which are trans in the native enzyme, resulted in no significant changes in the kinetic folding behavior. The conclusion from these experiments is that the 21-s p...Continue Reading

Citations

Sep 2, 2003·Current Opinion in Structural Biology·Ewan R G MainLynne Regan
Apr 16, 2009·Cellular and Molecular Life Sciences : CMLS·Tse Siang Kang, R Manjunatha Kini
Oct 4, 2005·Protein Science : a Publication of the Protein Society·David A SchultzRobert O Fox
Apr 23, 2015·Proceedings of the National Academy of Sciences of the United States of America·Thuy Phuong DaoDoug Barrick
Sep 17, 2008·Protein Science : a Publication of the Protein Society·Byoung-Chul Lee, Wouter D Hoff
Oct 30, 2007·Archives of Biochemistry and Biophysics·Ellen KlossDoug Barrick

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