Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed ''trigger sequence,'' that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation.autonomous folding unit ͉ protein folding ͉ trigger sequence ͉ leucine zipper ͉ ␣-helix A lthough coiled coils have been used traditionally as model systems for protein folding studies, the molecular basis of how they fold is still largely unknown. A need for a detailed understanding of coiled-coil folding is relevant, particularly in light of the important functions these structures play in almost all biological processes, as well as the considerable attention coiled coils have recently garnered in a wide range of applications, including basic research, nanomaterials, protein engineering, biotechnology, and medicine (1-13). Furthermore, understanding the folding mechanisms of coiled coils is of fundamental interest to experimentalists and theoreticians challenged by the question of how the sequence of a protein defines its specific 3D structure. Along these lines, we have been interested particularly in coiled-coil ''trigger sequences,'' which encode stable monomeric ␣-helices that are indispensable for coiled-coil formation (14-18). Although there are a few examples of synthetic peptides that fold either into heterodimers (19) or at conditions of extremes of pH (20) without an apparent trigger sequence, the ''trigger site'' concept is generally accepted because these short autonomous helical folding units are structurally and functionally conserved in a large number of native coiled-coil proteins (reviewed in refs. 21-24). Detailed knowledge of the properties of trigger sequences at the molecular level is therefore key to an understanding of coiled-coil formation and function in general.The parallel two-stranded leucine zipper of the yeast transcriptional activator GCN4 is the best-characterized coiled coil and thus represents an excellent paradigm for a comprehensive analysis of the roles of trigger sequences in coiled-coil folding. Our current understanding of the folding kinetics of the GCN4 leucine zipper is based on extensive stopped-flow studies and te...