Collagen triple-helix formation in all-trans chains proceeds by a nucleation/growth mechanism with a purely entropic barrier.
Collagen consists of repetitive Gly-Xaa-Yaa tripeptide units with proline and hydroxyproline frequently found in the Xaa and Yaa position, respectively. This sequence motif allows the formation of a highly regular triple helix that is stabilized by steric (entropic) restrictions in the constituent polyproline-II-helices and backbone hydrogen bonds between the three strands. Concentration-dependent association reactions and slow prolyl isomerization steps have been identified as major rate-limiting processes during collagen folding. To gain information on the dynamics of triple-helix formation in the absence of these slow reactions, we performed stopped-flow double-jump experiments on cross-linked fragments derived from human type III collagen. This technique allowed us to measure concentration-independent folding kinetics starting from unfolded chains with all peptide bonds in the trans conformation. The results show that triple-helix formation occurs with a rate constant of 113 +/- 20 s(-1) at 3.7 degrees C and is virtually independent of temperature, indicating a purely entropic barrier. Comparison of the effect of guanidinium chloride on folding kinetics and stability reveals that the rate-limiting step is represented by bringing 10 consecutive tripeptide units (3.3 per strand) into a triple-helical conformation. The following addition of tripeptide units occurs on a much faster time scale and cannot be observed experimentally. These results support an entropy-controlled zipper-like nucleation/growth mechanism for collagen triple-helix formation.
Bachmann
Annett
A
Department of Biophysical Chemistry, Biozentrum, University of Basel, CH 4056 Basel, Switzerland. annett.bachmann@unibas.ch
Kiefhaber
Thomas
T
Boudko
Sergei
S
Engel
Jürgen
J
Bächinger
Hans Peter
HP
D024061:Collagen Type III / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D019277:Entropy; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D017510:Protein Folding; D013696:Temperature
D024061:Collagen Type III; D004220:Disulfides; D019791:Guanidine