EFFECTS OF LANTHANIDE BINDING ON THE STABILITY OF DE-NOVO DESIGNED ALPHA-HELICAL COILED-COILS

Effects of La3+ ion binding on the stability of de novo designed two-s tranded a-helical coiled-coils were studied. The coiled-coils were com posed of two 35-residue polypeptide chains based on the ''native'' hep tad sequence Q(g)V(a)G(b)A(c)L(d)Q(e)K(f) and each contained a Cys res idue at position 2a to allow formation of an interchain disulfide brid ge. The effect of LaCl3 on the stability of five analogs containing tw o or three Glu substitutions per chain at heptad positions e and g was observed by urea denaturation at 20 degrees C. The analog E-2(15,20), in which Glu residues are involved in interhelical i to i' + 5 repuls ions, was stabilized relative to the control native peptide by additio n of 50 mM LaCl3 to the buffer, whereas two analogs, in which Glu resi dues do not interact, were destabilized. These results suggest that La Cl3 may preferentially stabilize the folded state of E-2(15,20) by the ''bridging'' of La3+ ions between two pairs of Glu residues usually i nvolved in interhelical repulsions. Two analogs designed to contain tw o La3+ binding sites composed of three Glu residues each show greater stabilization by LaCl3 than E,(15,20) in the disulfide-bridged form. T he apparent stabilization of E-2(15,20) by La3+ binding was not observ ed with either Ca2+ or Mg2+, indicating that the effect is specific fo r trivalent versus divalent cations. (C) Munksgaard 1997.