Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 359, Issue 2, Pages 289-298Publisher
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2006.02.071
Keywords
collagen; peptide; disulphide; integrin; helix
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Funding
- Medical Research Council [G0500707] Funding Source: Medline
- Medical Research Council [G0500707] Funding Source: researchfish
- MRC [G0500707] Funding Source: UKRI
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We report a rapid method to synthesize cystine cross-linked heterotrimeric collagenous peptides. They can be engineered to favour one particular axial alignment of the strands, called the register of the helix. Here, the sequence of the constituent peptides contains 18 residues of guest collagen type I sequence flanked by N and C-terminal (Gly-Pro-Pro)(5) host modules which ensure helicity. Further C-terminal residues include appropriately spaced cysteine residues and alanine to provide the necessary flexibility for helix formation. The cross-linking reaction and subsequent separation protocols have been designed for any inserted collagen sequence that does not contain a cysteine residue. Mass spectrometry and ion-exchange chromatography allow us to distinguish between different disulphide-bonded species and to monitor the formation of side-products. Starting peptide can be recovered simply from the reaction mixture by reduction and separation. Yields are typically 30%, working on a 10 mg scale. N-15-H-1 NMR and platelet adhesion studies show that the peptide heterotrimers presented here can reshuffle to cover all three axial registers. Less flexible spacers between the disulphide linkages and the helix will restrict each heterotrimer to one register only. (c) 2006 Elsevier Ltd. All rights reserved.
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