Abstract
Laminin, a large multidomain glycoprotein specific to basement membranes, is a heterotrimer with alpha, beta, and gamma chains held together in an alpha-helical coiled-coil structure. Synthetic peptides comprising two 51-mers (B1 and B2) from the beta 1 and gamma 1 subunits and a 55-mer (M) from alpha 2 were used to study the molecular mechanisms in laminin chain assembly. Using the synthetic peptides in various mixing experiments, the heterotrimer (B1-B2-M) was preferentially produced. The thermal stability of the heterotrimer increased dramatically (by approximately 20 degrees C) over that of the B1-B2 heterodimer as measured by circular dichroism (CD) spectroscopy. The B1-B1 homodimer (Tm = 60 degrees C) showed higher thermal stability when compared to B1-B2 and B2-B2 dimers. However, the B1 + B2 mixture produced principally the B1-B2 heterodimer. These results suggested that the preferential formations of heterodimer was regulated by kinetic interactions between each chain. The B2 and M peptides have many hydrophobic isoleucine residues which were replaced by leucines. These substitutions were predicted to favor an alpha-helical conformation and a higher propensity for zipper formation. B2L and ML, in which all isoleucine residues were replaced by leucine, showed significantly increased alpha-helicities. While B2L was able to form heterodimers and heterotrimers similar to B2, ML was not able to participate in heterotrimer formation as efficiently as the M peptide. The thermal stability of B1-B2L was comparable to that of B1-B2, but B2L and/or ML containing trimers showed lower thermal stability than B1-B2-M. These results suggest that the isoleucine residues in the alpha 2 and gamma 1 chains are critical for stabilizing the heteromeric triple-stranded coiled-coil structure.
Highlights
BI-B1 homodimer (T, = 60 “C) showed higher thermal stability when compared to B1-B2 and B2-B2 dimers
Thea-helical region has a similar size of higher propensity for zipper formationB. 2L and ML, in about 600 amino acids in each chain and consists of numerous which all isoleucine residues were replaced by leucine, repeats of a heptad sequence in which hydrophobic showed significantly increased a-helicities
Peptide Concentration-All peptide concentrations in this investigaarm of laminin-1, can be reconstitutedto a structure similarto tion were determined by amino acid analysis. the native molecule after denaturation 1 2 8 3 1 ) .More recently, Preparation of Disulfide-bonded DimerPeptides-Homodimers (Blwe have demonstrated that a short sequence of the carboxyl B1, B2-B2, and B2L-B2L) were prepared by air oxidation at a high end of each chain is required for specific assembly of laminin chains [32].In addition, studieswith site-directed mutagenesis have suggested that charged amino acid residues within these short carboxyl end sequences were important for specific formations of double- and triple-stranded coiled-coil structures concentration (5mg peptide/ml in 50 m sodium phosphate, pH 7.4)
Summary
Faculty of Pharmaceutical Sciences, Kyoto Univer- ethanol-aqueousbuffer. In contrast, thecorresponding regions sity, Sakyo-ku, Kyoto 606, Japan. The native molecule after denaturation 1 2 8 3 1 ) .More recently, Preparation of Disulfide-bonded DimerPeptides-Homodimers (Blwe have demonstrated that a short sequence of the carboxyl B1, B2-B2, and B2L-B2L) were prepared by air oxidation at a high end of each chain is required for specific assembly of laminin chains [32].In addition, studieswith site-directed mutagenesis have suggested that charged amino acid residues within these short carboxyl end sequences were important for specific formations of double- and triple-stranded coiled-coil structures concentration (5mg peptide/ml in 50 m sodium phosphate, pH 7.4). The products were applied onto a reversebinant proteins were used, we focused on the C-terminal 50 phase HPLC column, whereby the M peptide was dissociatferdom the amino acid region of the long arm, which is deemed to be the critical region of dimer and trimer assembly (Fig. 1).We synthesized peptide segments of the three subunitcsorresponding to this region and analyzed assembly of these peptides into heterotrimers under these conditions and dtihseulfide-bonded B1-B2 or Bl-B2L heterodimers were obtained in pure form. The a2 peptides (M and ML) and the dimer peptides
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