title: The effect of purity upon the triple-helical stability of collagenous peptides.
abstract: Collagen is the fundamental structural protein, comprising 25-35% of the total body protein, its rod-like triple helix providing support in many tissues. Our laboratory has synthesised 113 Toolkit peptides, each 63 residues long, covering the entirety of the homotrimeric helix sequence of collagen II and collagen III. These are used primarily to investigate protein-collagen interactions, from which biomedical applications are under development. Upon increasing the temperature of a Toolkit peptide solution, a novel low temperature transition (LTT) as well as a broadening of the helix unfolding higher temperature transition (HTT) was observed. Here, we hypothesized that unfolding of imperfect helices can account for the LTT. Peptides of various purities were isolated by HPLC or gel filtration, and their unfolding measured by polarimetry, CD, and DSC. The resulting temperature transitions were fitted to a kinetic unfolding equation, allowing comparison of the data, and explanation of the observed melting curve complexity as due to peptide imperfections. Finally, using a mathematical model, this data can be replicated by setting a parameter that quantifies the mutual stabilization conferred by helices on each side of a peptide defect within a triple helix.
authors.affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK. das55@cam.ac.uk
authors.forename: David A
authors.initials: DA
authors.lastname: Slatter
authors.forename: Dominique G
authors.initials: DG
authors.lastname: Bihan
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
mesh_terms: D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000737:chemistry* / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification*; D055550:Protein Stability; D017433:Protein Structure, Secondary
chemical_list: D010455:Peptides; D003094:Collagen