Rapid synthesis of a register-specific heterotrimeric type I collagen helix encompassing the integrin alpha2beta1 binding site.
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. 15N-1H 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.
Slatter
David A
DA
Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK. das@cam.ac.uk
Foley
Loraine A
LA
Peachey
Anthony R
AR
Nietlispach
Daniel
D
Farndale
Richard W
RW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D024042:Collagen Type I / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D038982:Integrin alpha2beta1 / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D010973:Platelet Adhesiveness; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D024042:Collagen Type I; D003432:Cross-Linking Reagents; D038982:Integrin alpha2beta1; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003553:Cystine