ABSTRACT
The redesign of protein-protein interactions is a stringent test of our understanding of molecular recognition and specificity. Previously we engineered a modest specificity switch into the colicin E7 DNase-Im7 immunity protein complex by identifying mutations that are disruptive in the native complex, but can be compensated by mutations on the interacting partner. Here we extend the approach by systematically sampling alternate rigid body orientations to optimize the interactions in a binding mode specific manner. Using this protocol we designed a de novo hydrogen bond network at the DNase-immunity protein interface and confirmed the design with X-ray crystallographic analysis. Subsequent design of the second shell of interactions guided by insights from the crystal structure on tightly bound water molecules, conformational strain, and packing defects yielded new binding partners that exhibited specificities of at least 300-fold between the cognate and the non-cognate complexes. This multi-step approach should be applicable to the design of polar protein-protein interactions and contribute to the re-engineering of regulatory networks mediated by protein-protein interactions.
Subject(s)
Computational Biology , Drug Design , Protein Interaction Mapping , Bacterial Proteins/chemical synthesis , Bacterial Proteins/genetics , Binding Sites , Carrier Proteins/chemical synthesis , Carrier Proteins/genetics , Colicins/chemical synthesis , Colicins/genetics , Crystallography, X-Ray , Escherichia coli Proteins/chemical synthesis , Escherichia coli Proteins/genetics , Hydrogen Bonding , Models, MolecularABSTRACT
To establish a system to address questions concerning the influence of glycosylation on protein folding pathways, we have developed a semisynthetic route toward the immunity protein Im7. This fourhelix protein has been used extensively as model protein for folding studies. Native chemical ligation (NCL) affords an N-linked chitobiose glycoprotein analogue of Im7 with an Ala29Cys mutation. The semisynthetic approach relies on the solid-phase peptide synthesis (SPPS) of N-terminal thioesters (including helix I), in glycosylated or unglycosylated form, in combination with the expression of the C-terminal fragment of Im7 (containing helices II-IV). Detailed kinetic and thermodynamic analysis of the protein folding behavior reveals that semisynthetic Im7 analogues are well suited for protein folding studies and that the folding mechanism of the glycoprotein of this Im7 variant is not significantly altered over the unglycosylated analogue.
