Designed Coiled Coils promote folding of a recombinant bacterial collagen

A Yoshimura, JM Fletcher, Z Yu, AV Persikov, GJ Bartlett, AL Boyle, TL Vincent, DN Woolfson, B Brodsky

Research output: Contribution to journalArticle (Academic Journal)peer-review

24 Citations (Scopus)

Abstract

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.
Translated title of the contributionDesigned Coiled Coils promote folding of a recombinant bacterial collagen
Original languageEnglish
Pages (from-to)17512 - 17520
Number of pages9
JournalJournal of Biological Chemistry
Volume286 (20)
DOIs
Publication statusPublished - May 2011

Bibliographical note

Other: Supplemental material can be found at: http:\\www.jcb.org/content/suppl/2011/03/28/M110.217364.DC1.html

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