The Relationship between Effective Molarity and Affinity Governs Rate Enhancements in Tethered Kinase-Substrate Reactions.

Scaffold proteins are thought to accelerate protein phosphorylation reactions by tethering kinases and substrates together, but there is little quantitative data on their functional effects. To assess the contribution of tethering to kinase reactivity, we compared intramolecular and intermolecular kinase reactions in a minimal model system. We found that tethering can enhance reaction rates in a flexible tethered kinase system and that the magnitude of the effect is sensitive to the structure of the tether. The largest effective molarity we obtained was ∼0.08 µM, which is much lower than the effects observed in small molecule model systems and other tethered protein reactions. We further demonstrated that the tethered intramolecular reaction only makes a significant contribution to the observed rates when the scaffolded complex assembles at concentrations below the effective molarity. These findings provide a quantitative framework that can be applied to understand endogenous protein scaffolds and engineer synthetic networks.

Designed Proteins as Novel Imaging Reagents in Living Escherichia coli.

Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co-expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs.

A designed repeat protein as an affinity capture reagent.

Repeat proteins are an attractive target for protein engineering and design. We have focused our attention on the design and engineering of one particular class: tetratricopeptide repeat (TPR) proteins. In previous work, we have shown that the structure and stability of TPR proteins can be manipulated in a rational fashion [Cortajarena (2011) Prot. Sci. 20: , 1042-1047; Main (2003) Structure 11: , 497-508]. Building on those studies, we have designed and characterized a number of different peptide-binding TPR modules and we have also assembled these modules into supramolecular arrays [Cortajarena (2009) ACS Chem. Biol. 5: , 545-552; Cortajarena (2008) ACS Chem. Biol. 3: , 161-166; Jackrel (2009) Prot. Sci. 18: , 762-774; Kajander (2007) Acta Crystallogr. D Biol. Crystallogr. 63: , 800-811]. Here we focus on the development of one such TPR-peptide interaction for a practical application, affinity purification. We illustrate the general utility of our designed protein interaction. Furthermore, this example highlights how basic research on protein-peptide interactions can lead to the development of novel reagents with important practical applications.

NextGen protein design.

Protein engineering is at an exciting stage because designed protein-protein interactions are being used in many applications. For instance, three designed proteins are now in clinical trials. Although there have been many successes over the last decade, protein engineering still faces numerous challenges. Often, designs do not work as anticipated and they still require substantial redesign. The present review focuses on the successes, the challenges and the limitations of rational protein design today.

The Scaffold Protein Axin Promotes Signaling Specificity within the Wnt Pathway by Suppressing Competing Kinase Reactions.

Scaffold proteins are thought to promote signaling specificity by accelerating reactions between bound kinase and substrate proteins. To test the long-standing hypothesis that the scaffold protein Axin accelerates glycogen synthase kinase 3ß (GSK3ß)-mediated phosphorylation of ß-catenin in the Wnt signaling network, we measured GSK3ß reaction rates with multiple substrates in a minimal, biochemically reconstituted system. We observed an unexpectedly small, ∼2-fold Axin-mediated rate increase for the ß-catenin reaction when measured in isolation. In contrast, when both ß-catenin and non-Wnt pathway substrates are present, Axin accelerates the ß-catenin reaction by preventing competition with alternative substrates. At high competitor concentrations, Axin produces >10-fold rate effects. Thus, while Axin alone does not markedly accelerate the ß-catenin reaction, in physiological settings where multiple GSK3ß substrates are present, Axin may promote signaling specificity by suppressing interactions with competing, non-Wnt pathway targets. This mechanism for scaffold-mediated control of competition enables a shared kinase to perform distinct functions in multiple signaling networks.

Design of Protein-Peptide Interaction Modules for Assembling Supramolecular Structures in Vivo and in Vitro.

Synthetic biology and protein origami both require protein building blocks that behave in a reliable, predictable fashion. In particular, we require protein interaction modules with known specificity and affinity. Here, we describe three designed TRAP (Tetratricopeptide Repeat Affinity Protein)-peptide interaction pairs that are functional in vivo. We show that each TRAP binds to its cognate peptide and exhibits low cross-reactivity with the peptides bound by the other TRAPs. In addition, we demonstrate that the TRAP-peptide interactions are functional in many cellular contexts. In extensions of these designs, we show that the binding affinity of a TRAP-peptide pair can be systematically varied. The TRAP-peptide pairs we present thus represent a powerful set of new building blocks that are suitable for a variety of applications.

White and green screening with circular polymerase extension cloning for easy and reliable cloning.

Cloning is an essential prerequisite to test protein design and engineering ideas. However, it is often time consuming, unreliable, and therefore frustrating. Here, we present a streamlined cloning strategy that incorporates a powerful white and green screening protocol to identify colonies with inserts. We use circular polymerase extension cloning, which is both ligation and sequence independent. Furthermore, our entire procedure requires only three quick steps and one enzyme making it easy to use, inexpensive, and tractable. We anticipate that this method will be particularly useful for protein engineers who frequently subclone or make focused deletion, insertion, or substitution libraries.