Synthesis and conformation of the trimeric coiled-coil segment of laminin

A disulfide linked 95-mer parallel hetero-trimeric active site segment of laminin was designed and synthesised. The three subunits, A (32-mer), B1 (30-mer) and B2 (33-mer), were prepared by Boc-based solid-phase peptide synthesis involving a two-step trimethylsilyl bromide-thioanisole and HF deprotection procedure. The interlinking of the three subunits was accomplished by the stepwise selective formation of two disulfide bridges using air-oxidation and thallium (III) trifluoroacetate oxidation. The conformations of the synthetic peptides were studied by circular dichroism (CD) spectroscopy, showing that the hetero-dimer, B1-B2, one of the homo-dimers, B1-B1, and the trimer are 30 to 40% in the alpha-helical conformation in aqueous buffer. Variable temperature CD studies demonstrated that the trimer is considerably more stable (melting temperature (Tm) = 61 degrees) than the hetero-dimer, B1-B2 (Tm = 36 degrees).