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-stranded alpha-helical coiled-coils were studied. The coiled-coils were composed of two 35-residue polypeptide chains based on the "native" heptad sequence Q(g)V(a)G(b)A(c)L(d)Q(e)K(f) and each contained a Cys residue at position 2a to allow formation of an interchain disulfide bridge. The effect of LaCl3 on the stability of five analogs containing two or three Glu substitutions per chain at heptad positions e and g was observed by urea denaturation at 20 degrees C. The analog E2(15,20), in which Glu residues are involved in interhelical i to i' + 5 repulsions, was stabilized relative to the control native peptide by addition of 50 mM LaCl3 to the buffer, whereas two analogs, in which Glu residues do not interact, were destabilized. These results suggest that LaCl3 may preferentially stabilize the folded state of E2(15,20) by the "bridging" of La3+ ions between two pairs of Glu residues usually involved in interhelical repulsions. Two analogs designed to contain two La3+ binding sites composed of three Glu residues each show greater stabilization by LaCl3 than E2(15,20) in the disulfide-bridged form. The apparent stabilization of E2(15,20) by La3+ binding was not observed with either Ca2+ or Mg2+, indicating that the effect is specific for trivalent versus divalent cations.
Kohn
W D
WD
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
Hodges
R S
RS
D000595:Amino Acid Sequence; D002122:Calcium Chloride / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D015636:Magnesium Chloride / Q000737:chemistry; D008674:Metals, Rare Earth / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D008674:Metals, Rare Earth; D010455:Peptides; D015636:Magnesium Chloride; D014508:Urea; D002122:Calcium Chloride