The long arm of laminin in which three polypeptide chains alpha, beta, and gamma are assembled in an alpha-helical coiled-coil structure is stabilized by non-covalent interactions and disulfide bridges. The stabilizing role of the disulfide linkage between the beta and gamma-chains at the C-terminal region of the assembly domain was investigated with about 100-residue long recombinant fragments. Circular dichroism spectra and electron micrographs were identical for linked and non-linked species and indicated two-stranded coiled-coil structures with about 100% alpha-helicity at 20 degrees C. Thermal transition profiles revealed an increase of the melting temperature from 42 degrees C to 60.4 degrees C upon disulfide formation at a chain concentration of 25 microM. The enthalpy of interaction was identical for the two species but the negative entropy involved in joining the two chains was reduced by the disulfide bonds. At chain concentrations of 10 microM the Gibbs free energy delta G was by 17.5 kJ/mol more negative for the disulfide-linked than for the unlinked chains. Because of the concentration dependence of the entropy of the non-linked chains, this difference decreased with increasing concentration and, by extrapolation at chain concentrations of 10 mM, the stability of both structures would be the same. As a competing reaction, beta-chains associated to four-stranded bundles which probably consist of pairs of two-stranded coiled-coils. After disulfide formation a biphasic transition curve was observed which indicated two different ways of connecting the chains in the bundle. Study holds ProTherm entries: 7491, 7492 Extra Details: laminin; alpha-helical coiled-coil; recombinant fragments;,disulfide linkage; circular dichroism
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:34 p.m.
|Number of data points||5|
|Proteins||Laminin subunit alpha-2 ; Laminin subunit alpha-2|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence APLAANAESGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTRPTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAIYDAEIPGHMCNGQWHKVTAKKIKNRLELVVDGNQVDAQSPNSASTSADTNDPVFVGGFPGGLNQFGLTTNIRFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPTT|