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).
Nomizu
M
M
Laboratory of Medicinal Chemistry, DTP, DCT, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Utani
A
A
Shiraishi
N
N
Yamada
Y
Y
Roller
P P
PP
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D007797:Laminin / Q000138:chemical synthesis* / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D004220:Disulfides; D007797:Laminin; D046911:Macromolecular Substances