Electrostatic Interactions Control the Parallel and Antiparallel Orientation of .alpha.-Helical Chains in Two-Stranded .alpha.-Helical Coiled-Coils

Abstract

The role of interchain electrostatic interactions in orientating alpha-helical chains to form two-stranded parallel and antiparallel coiled-coils has been investigated. Four disulfide-bridged coiled-coils were designed: parallel coiled-coils with interchain electrostatic attractions (P/A) and repulsions (P/R) and antiparallel coiled-coils with interchain electrostatic attractions (AP/A) and repulsions (AP/R). These coiled-coils were made by air oxidation of two 35-residue peptides with the appropriate heptad repeat (LaEbAcLdEeGfKg or LaAbEcLdKeGfEg) to give the desired interchain electrostatic interactions, and the appropriate position of the cysteine residue (C2 or C33) to give the desired chain orientation. The coiled-coils were characterized by circular dichroism spectroscopy, and their stabilities were assessed by guanidine hydrochloride and urea denaturations. The results indicated that the favored chain orientation, that is, the major disulfide-bridged product formed under benign conditions, was the one that provides interchain electrostatic attractions between oppositely-charged amino acid residues in the e-g' and g-e' positions of the parallel coiled-coil and the g-g' and e-e' positions in the antiparallel coiled-coil. When the electrostatic interactions were similar, the antiparallel coiled-coils were more stable than the parallel coiled-coils. However, the overall stability of the coiled-coils was either increased by interchain electrostatic attractions or decreased by interchain electrostatic repulsions, as determined by urea denaturation. Thus, the order of overall stability of these coiled-coils was AP/A > P/A > AP/R > P/R. This study demonstrates the importance of interchain electrostatic interactions in determining the parallel or antiparallel orientation of alpha-helical chains in two-stranded coiled-coils.

Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions

“…These results strongly support our earlier contention that the difference in the estimates of their stabilities was due to the ionic property of GdnHCl and its absence in urea (Monera et al, 1993(Monera et al, , 1994. It is well understood that GdnHCl is a salt and, therefore, expected to ionize in aqueous solution.…”

“…2B). Their CD profiles in benign medium showed that the 2 minima at 220 and 208 nm had a molar ellipticity ratio of -1, which had been previously attributed to coiled-coil structures (Lau et al, 1984;Zhou et al, 1992aZhou et al, , 1992bZhu et al, 1992;Monera et al, 1993Monera et al, , 1994. In the presence of TFE, which disrupts hydrophobic interactions, the CD profiles became more characteristic of single-stranded a-helices.…”

“…For both denaturants, a good correlation has been observed between the number of denaturant molecules bound and a summation of one-half the number of peptide bonds plus the number of aromatic amino acid residues (Lee & Timasheff, 1974;Prakash et al, 1981). Although the above interpretation is generally true, there seems to be a disagreement as to whether the estimates of protein stability are dependent Monera et al, 1993Monera et al, , 1994 or independent (Greene & Pace, 1974;Ahmad & Bigelow, 1982;Bowie & Sauer, 1989;Pace et al, 1990) of the denaturant used.…”

“…More recently, Pace et al (1990) hinted that the much higher ionic strength of the GdnHCl solutions would be expected to suppress effects due to electrostatic interactions among charged groups in proteins. Incidentally, in the course of studying coiled-coil peptides, we observed that 2 coiled-coil analogs that had similar hydrophobic packing but with different electrostatic interactions had identical [GdnHCl]l/2 values, but greatly differed in rea]^/^ values (Monera et al, 1993(Monera et al, , 1994. Also, when the ratio of GdnHCl to urea in the denaturant mixture was gradually changed, this was accompanied by a stepwise shift in [ d e n a t~r a n t ]~,~ values, the direction of which was dependent on the type of electrostatic interactions in the coiled-coil (Monera et al, 1993).…”

Computational analysis of residue contributions to coiled‐coil topology

“…Thus, a design where the e and g positions have opposite polarities would result in interchain electrostatic attraction in the parallel topology, whereas residues with the same polarity would stabilize the antiparallel orientation. 14,15 The importance of electrostatic interactions was also evident in the case of the C-terminal domain from bovine inhibitor protein of F1 ATPase, which forms an antiparallel coiled-coil dimer but switches to tetramer above pH 6.5. 16 Another factor that affects the alignment of the helices is packing.…”