Hydrogen Bond Surrogate Stabilization of β‑Hairpins

Peptide secondary and tertiary structure motifs frequently serve as inspiration for the development of protein–protein interaction (PPI) inhibitors. While a wide variety of strategies have been used to stabilize or imitate α-helices, similar strategies for β-sheet stabilization are more limited. Synthetic scaffolds that stabilize reverse turns and cross-strand interactions have provided important insights into β-sheet stability and folding. However, these templates occupy regions of the β-sheet that might impact the β-sheet’s ability to bind at a PPI interface. Here, we present the hydrogen bond surrogate (HBS) approach for stabilization of β-hairpin peptides. The HBS linkage replaces a cross-strand hydrogen bond with a covalent linkage, conferring significant conformational and proteolytic resistance. Importantly, this approach introduces the stabilizing linkage in the buried β-sheet interior, retains all side chains for further functionalization, and allows efficient solid-phase macrocyclization. We anticipate that HBS stabilization of PPI β-sheets will enhance the development of β-sheet PPI inhibitors and expand the repertoire of druggable PPIs

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones

Designed Phosphoprotein Recognition in <i>Escherichia coli</i>

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide–protein interactions in <i>Escherichia coli</i>. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide <i>in vitro</i>. We then combine <i>in vivo</i> site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide–protein interaction specificity in <i>E. coli</i>. This <i>in vivo</i> strategy for detecting and characterizing phosphopeptide–protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones