PMID- 11926834
OWN - NLM
STAT- MEDLINE
DCOM- 20020517
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 41
IP  - 14
DP  - 2002 Apr 9
TI  - Folding kinetics of the protein pectate lyase C reveal fast-forming intermediates 
      and slow proline isomerization.
PG  - 4713-23
AB  - 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.
FAU - Kamen, Douglas E
AU  - Kamen DE
AD  - Department of Biochemistry and Molecular Biology, Colorado State University, Fort 
      Collins, Colorado 80523, USA.
FAU - Woody, Robert W
AU  - Woody RW
LA  - eng
GR  - GM-22994/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Isoenzymes)
RN  - 9DLQ4CIU6V (Proline)
RN  - EC 4.2.2.- (Polysaccharide-Lyases)
RN  - EC 4.2.2.2 (pectate lyase)
SB  - IM
MH  - Circular Dichroism
MH  - Crystallography, X-Ray
MH  - Isoenzymes/*chemistry/metabolism
MH  - Isomerism
MH  - Kinetics
MH  - Models, Molecular
MH  - Polysaccharide-Lyases/*chemistry/metabolism
MH  - Proline/chemistry/*metabolism
MH  - Protein Conformation
MH  - Protein Denaturation
MH  - Protein Folding
MH  - Protein Renaturation
MH  - Protein Structure, Secondary
MH  - Spectrometry, Fluorescence
MH  - Spectrophotometry, Ultraviolet
EDAT- 2002/04/03 10:00
MHDA- 2002/05/23 10:01
CRDT- 2002/04/03 10:00
PHST- 2002/04/03 10:00 [pubmed]
PHST- 2002/05/23 10:01 [medline]
PHST- 2002/04/03 10:00 [entrez]
AID - bi0115129 [pii]
AID - 10.1021/bi0115129 [doi]
PST - ppublish
SO  - Biochemistry. 2002 Apr 9;41(14):4713-23. doi: 10.1021/bi0115129.