Selective chain recognition in the C-terminal alpha-helical coiled-coil region of laminin.
Recombinant fragments alpha, beta, and gamma were prepared comprising about 100 C-terminal residues of the corresponding polypeptide chains in the three-stranded alpha-helical coiled-coil domain of laminin. Circular dichroism spectra, thermal transition profiles, non-denaturing gels, analytical ultracentrifugation, and calorimetry indicated alpha-helicity and high thermal stabilities for the beta gamma heterodimer and homoassociates of beta. Very little or no coiled-coil formation was found for alpha and gamma. The thermal melting profiles and their concentration dependencies were quantitatively described by a two-state mechanism in which two unfolded chains combine to a fully alpha-helical dimer. For the beta gamma dimer the melting temperature was Tm = 42 degrees C at a chain concentration of 25 microM in 5 mM sodium phosphate buffer (pH 7.4). Addition of 100 mM NaCl decreased the Tm slightly but the relative stability of beta gamma and beta beta coiled-coils was not significantly changed, indicating that electrostatic interactions alone are not responsible for chain selection. Upon addition of 1 M urea the Tm value dropped by about 10 degrees C. The enthalpy changes for the formation of the coiled-coil were delta H degrees = -304(+/- 30) kJ/mol for the beta gamma heterodimer and -198(+/- 20) kJ/mol for the beta-homoassociates. Gibbs free energies and entropies amounted to delta G degrees = -42.8 kJ and delta S degrees = -876 J/mol K for the heteroassembly and -37.8 kJ/mol and -537 J/mol K for the homoassembly of beta. This low preference for heteroassociation of the fragment is smaller than the chain selectivity observed for larger fragments and intact laminin. Deletion of ten residues from the C-terminal region of the gamma-fragment which were recently reported as an essential assembly-site was not sufficient to abolish heteroassociation. Interaction of alpha-fragment with double-stranded beta gamma coiled-coils reflected the formation of a three-stranded coiled-coil in laminin but for the small recombinant fragments association between alpha and beta-homoassociates was also observed. The C-terminal 100 residues in the coiled-coil domain are therefore not alone responsible for the high specificity of chain selection in laminin.
Kammerer
R A
RA
Department of Biophysical Chemistry, Biozentrum, Basel, Switzerland.
Antonsson
P
P
Schulthess
T
T
Fauser
C
C
Engel
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002151:Calorimetry; D007700:Kinetics; D007797:Laminin / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion / Q000502:physiology; D013816:Thermodynamics
D007797:Laminin; D010446:Peptide Fragments; D011994:Recombinant Proteins