Folding kinetics of the protein pectate lyase C reveal fast-forming intermediates and slow proline isomerization

Biochemistry
D E Kamen, R W Woody

Abstract

Pectate lyase C (pelC) is a member of the class of proteins that possess a parallel beta-helix folding motif. A study of the kinetic folding mechanism is presented in this report. Kinetic circular dichroism (CD) and fluorescence have been used to observe changes in the structure of pelC as a function of time upon folding and unfolding. Three folding phases are observed with far-UV CD and four phases are observed with near-UV CD. The two slowest phases have relaxation times on the order of 21 and 46 s in aqueous buffer. Double-jump refolding assays and the measured activation enthalpies (16.0 and 21.2 kcal/mol for the respective slow phases) suggest that these two phases are the result of the slow cis-trans isomerization of prolyl-peptide bonds. We have determined that the earliest observed folding phase involves the formation of most, if not all, of the secondary structure with a relaxation time of 0.25 s. We also observed a phase by near-UV CD on the order of 0.25 s. This suggests that along with the appearance of secondary structure, some tertiary contacts are made. There is one kinetic phase observed in the near-UV CD and fluorescence that has no corresponding far-UV CD phase. This occurs with a relaxation time of 1.1 s. The...Continue Reading

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Citations

Mar 9, 2004·Journal of Molecular Biology·Alexander FreibergRobert Seckler
Aug 21, 2003·Journal of Molecular Biology·Andreas Martin, Franz X Schmid
Sep 2, 2003·Current Opinion in Structural Biology·Ewan R G MainLynne Regan
Dec 17, 2014·Protein Expression and Purification·Sandeep KumarRamesh Chander Kuhad
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 23, 2009·Proteins·Mirco Junker, Patricia L Clark
Oct 30, 2007·Archives of Biochemistry and Biophysics·Ellen KlossDoug Barrick
Sep 24, 2005·The Journal of Biological Chemistry·Liyan ZhangGunilla Olivecrona
Oct 13, 2009·Applied and Environmental Microbiology·Jonathan CaspiEdward A Bayer
Jul 10, 2003·Chemical Reviews·Christophe Dugave, Luc Demange

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