2011
DOI: 10.1074/jbc.m110.217364
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Designed Coiled Coils Promote Folding of a Recombinant Bacterial Collagen

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 ␣-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, ␣-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra … Show more

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Cited by 33 publications
(25 citation statements)
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“…The PTE 3 was engineered by combining the phosphotriesterase gene from Brevundimonas diminuta [24,25] with the collagen-like triple helix domain and the trimerization V domain from Streptococcus pyogenes [26,27] referred to as PTE 3 . The PTE 3 cassette was excised from a maintenance plasmid and cloned to the bacterial expression vector pET28 (Stratagene) via a NcoI/NotI restriction enzyme digest.…”
Section: Phosphotriesterase Trimer Enzyme Preparationmentioning
confidence: 99%
“…The PTE 3 was engineered by combining the phosphotriesterase gene from Brevundimonas diminuta [24,25] with the collagen-like triple helix domain and the trimerization V domain from Streptococcus pyogenes [26,27] referred to as PTE 3 . The PTE 3 cassette was excised from a maintenance plasmid and cloned to the bacterial expression vector pET28 (Stratagene) via a NcoI/NotI restriction enzyme digest.…”
Section: Phosphotriesterase Trimer Enzyme Preparationmentioning
confidence: 99%
“…In a more recent study, Scl2-V was replaced with a hyperstable three-stranded coiled-coil, either at the N-terminus or the C-terminus of the triple-helix. The chimeric proteins retain their distinctive melting temperatures, but the rate of refolding was faster when the coiled-coil was at C-terminus (Yoshizumi et al 2011). …”
Section: Characterization and Manipulation Of Trimerization Domainmentioning
confidence: 99%
“…[19][20][21][22] Together, such heuristics have been used to design a suite of coiled-coil components, 8,[23][24][25] which have been used as modular building blocks in various synthetic-biology applications. [26][27][28][29][30] The above said, the a/d interfaces are not the exclusive province of hydrophobic residues; indeed, ≈1/4 of residues at these interfaces of structurally defined coiled coils are polar. 2,4,10 In many instances, these residues are conserved through evolution, 31 and likely play vital roles in specifying coiled-coil oligomeric state and function.…”
Section: Introductionmentioning
confidence: 95%