Collagen triple-helix formation in all-trans chains proceeds by a nucleation/growth mechanism with a purely entropic barrier
- *Department of Biophysical Chemistry, Biozentrum, University of Basel, CH 4056 Basel, Switzerland; and §Research Center, Shriners Hospital for Children, Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239
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Edited by Robert L. Baldwin, Stanford University Medical Center, Stanford, CA, and approved August 16, 2005 (received for review June 18, 2005)
Abstract
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°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.
Footnotes
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↵ † To whom correspondence may be addressed. E-mail: annett.bachmann{at}unibas.ch or hpb{at}shcc.org.
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↵ ‡ Present address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2054.
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Author contributions: A.B., J.E., and H.P.B. designed research; A.B., S.B., and H.P.B. performed research; S.B. and H.P.B. contributed new reagents/analytic tools; and A.B. and T.K. analyzed data and wrote the paper.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: Hyp, 4-hydroxyprolines; GdmCl, guanidinium chloride.
- Copyright © 2005, The National Academy of Sciences
