Are trigger sequences essential in the folding of two-stranded alpha-helical coiled-coils?

The amino acid residues comprising the interface between strands of the coi led-coil motif are usually hydrophobic and make a major contribution to coi led-coil folding and stability. However, in some cases the presence of exce llent hydrophobic residues at the coiled-coil interface is insufficient for folding. It has been proposed that a "consensus trigger sequence" exists t hat is necessary within the coiled-coil domains of various proteins to trig ger folding. Therefore, in this study we designed a 31-residue hybrid seque nce based on sequences from the two-stranded parallel coiled-coil domains o f the yeast transcriptional activator GCN4 and the actin-bundling protein D ictyostelium discoideum cortexillin I. The hybrid and its analogs were stud ied by CD spectroscopy and analytical ultracentrifugation. The hybrid had s table residues in the core "a" and "d" positions in the 3-4 hydrophobic rep eat, denoted (abcdefg)(n), but did not have a consensus trigger sequence an d did not possess appreciable secondary structure as determined by CD spect roscopy. The substitutions in the parent peptide were introduced at positio ns other than "a" and "d", altering a variety of interactions including alp ha -helical propensity, interchain and intrachain electrostatics, and hydro phobicity. Although the substitutions did not bring the overall sequence in closer agreement to the consensus trigger sequence, they increased coiled- coil folding and stability. Therefore, our results suggest that the combina tion of stabilizing effects along a protein sequence is a more general indi cator of protein folding in coiled-coils than the identification of a speci fic trigger sequence. We propose that surpassing a critical threshold stabi lity value using any type or combination of stabilizing effects will allow coiled-coils to fold, in the absence of a specific trigger sequence per se. (C) 2001 Academic Press.