ORIENTATION, POSITIONAL, ADDITIVITY, AND OLIGOMERIZATION-STATE EFFECTS OF INTERHELICAL ION-PAIRS IN ALPHA-HELICAL COILED-COILS

The role of interhelical g-e' ion pairs in the dimerization specificit y and stability of alpha-helical coiled-coils is highly controversial. Synthetic 35-residue coiled-coils based on the heptad repeat Q(g)V(a) G(b)A(c)L(d)Q(e)K(f) were used to investigate the effect of orientatio n of interhelical ion pairs between lysine and glutamic acid residues on coiled-coil stability. Stability was estimated from urea denaturati on at 20 degrees C, monitoring unfolding with circular-dichroism spect roscopy. Double mutant cycles were employed to estimate the net intera ction energy, Delta Delta G(u)(int), for the two orientations of the i on pair; E-e-K-g and K-e-E-g. Delta Delta G(u)(int) was found to be ab out 1.4-fold higher for the E-e-K-g orientation in a coiled-coil conta ining an N-terminal disulfide bridge, Delta Delta G(u)(int) value was similar whether obtained from the middle heptad or averaged over all f ive heptads of the coiled-coil, suggesting that ion pairs contribute a dditively to coiled-coil stability. The effect of uncompensated charge s was also illustrated by single substitutions of Gin with either Lys or Glu, resulting in Lys-Gln or Glu-Gln g-e' pairs. These substitution s were found to be twice as destabilizing at position g as at position e, and Lys was about twice as destabilizing as Glu at both positions e and g. In the absence of an interhelical disulfide bridge, Glu and L ys substitutions in the middle heptad were equally destabilizing at po sitions e and g (Lys continued to be more destabilizing than Glu) and the Delta Delta G(u)(int) value for Lys-Glu ion pairs was not orientat ion dependent. These and previous results suggest the non-covalently-l inked synthetic coiled-coils behave as molten globules, whereas a disu lfide-bridge may ''lock in'' the structural differences between positi ons of the heptad repeat. Interhelical Lys-Glu ion pairs in either ori entation promoted the formation of trimeric coiled-coils tin the absen ce of a disulfide bridge) while Gln-Gln g-e' interactions led to dimer formation. The results support a role for g-e' ionic attractions in c ontrolling coiled-coil specificity, stability and oligomerization stat e, possibly through effects on the side-chain packing at the subunit i nterface. (C) 1998 Academic Press.