Signaling pathways depend on regulatory protein-protein interactions; controlling these interactions in cells has important applications for reengineering biological functions. As many regulatory proteins are modular, considerable progress in engineering signaling circuits has been made by recombining commonly occurring domains. Our ability to predictably engineer cellular functions, however, is constrained by complex crosstalk observed in naturally occurring domains. Here we demonstrate a strategy for improving and simplifying protein network engineering: using computational design to create orthogonal (non-crossreacting) protein-protein interfaces. We validated the design of the interface between a key signaling protein, the GTPase Cdc42, and its activator, Intersectin, biochemically and by solving the crystal structure of the engineered complex. The designed GTPase (orthoCdc42) is activated exclusively by its engineered cognate partner (orthoIntersectin), but maintains the ability to interface with other GTPase signaling circuit components in vitro. In mammalian cells, orthoCdc42 activity can be regulated by orthoIntersectin, but not wild-type Intersectin, showing that the designed interaction can trigger complex processes. Computational design of protein interfaces thus promises to provide specific components that facilitate the predictable engineering of cellular functions.computational modeling and design | signal transduction | synthetic biology

Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of R-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33;Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641. We have made site-specific substitutions in the N-and C-cap motifs of the 5-helix protein monomeric λ repressor (λ 6-85 ) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly contextdependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.The native and denatured conformational ensembles involved in the process of protein folding are important in proper cellular function. If the native state is the functional species, then that function is lost during any time spent in the unfolded state or in the folding process. The denatured or unfolded ensemble of structures may be more susceptible to protein turnover through cellular degradation pathways. Additionally, proper cellular localization and protein trafficking is often coupled to the folding process. A detailed understanding of the medically important process of protein misfolding and aggregation requires better molecular characterization of kinetic folding mechanisms.Each residue in a protein does not have an equal role in the folding process. Several studies have shown that often only a small subset of residues attain nativelike structure in the transition state (3-6). In this paper, we consider the role of helix termini in protein folding by determining the kinetic consequences of site-specific substitutions in helix N-and C-cap structural motifs.Helix N-and C-cap residues are found at the beginning and end of helices, ending the regular i to i + 4 hydrogen bond pattern ...

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