title: Folding kinetics of the protein pectate lyase C reveal fast-forming intermediates and slow proline isomerization.
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 temperature dependence of the natural log of the folding rate constant suggests that folding occurs via a sequential mechanism in which an on-pathway intermediate in rapid equilibrium with the unfolded protein is present. Semiempirical CD calculations support the idea that the beta-helix region of pelC forms in the fast kinetic phase, yielding near-native secondary and tertiary structures in that region. This is followed by the slower formation of the loop regions connecting individual strands of the beta-helix.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, USA.
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Kamen
authors.forename: Robert W
authors.initials: RW
authors.lastname: Woody
mesh_terms: D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000378:metabolism; D011392:Proline / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007527:Isoenzymes; D011392:Proline; D011133:Polysaccharide-Lyases; C023911:pectate lyase