Strategies for Engineering and Rewiring Kinase Regulation.

Eukaryotic protein kinases (EPKs) catalyze the transfer of a phosphate group onto another protein in response to appropriate regulatory cues. In doing so, they provide a primary means for cellular information transfer. Consequently, EPKs play crucial roles in cell differentiation and cell-cycle progression, and kinase dysregulation is associated with numerous disease phenotypes including cancer.

Engineering allosteric regulation in protein kinases.

Phosphoregulation, in which the addition of a negatively charged phosphate group modulates protein activity, enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data revealed a set of spatially distributed "hotspots" that might have coevolved with the active site and preferentially modulated kinase activity.

An evolution-based strategy for engineering allosteric regulation.

Allosteric regulation provides a way to control protein activity at the time scale of milliseconds to seconds inside the cell. An ability to engineer synthetic allosteric systems would be of practical utility for the development of novel biosensors, creation of synthetic cell signaling pathways, and design of small molecule pharmaceuticals with regulatory impact. To this end, we outline a general approach-termed rational engineering of allostery at conserved hotspots (REACH)-to introduce novel regulation into a protein of interest by exploiting latent allostery that has been hard-wired by evolution into its structure.

Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae.

D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S.

Single-cell measurements of enzyme levels as a predictive tool for cellular fates during organic acid production.

Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity.

Assigning quantitative function to post-translational modifications reveals multiple sites of phosphorylation that tune yeast pheromone signaling output.

Cell signaling systems transmit information by post-translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge.

Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range.

Although the proteins comprising many signaling systems are known, less is known about their numbers per cell. Existing measurements often vary by more than 10-fold. Here, we devised improved quantification methods to measure protein abundances in the Saccharomyces cerevisiae pheromone response pathway, an archetypical signaling system.

Reagents for investigating MAPK signalling in model yeast species.

Here we present a set of resources (bacterial expression plasmids and antibodies) for the interrogation of proteins involved in yeast MAPK signalling. We constructed bacterial protein expression plasmids for 25 proteins involved in MAPK signalling in budding yeast. From these constructs we expressed and purified proteins and generated rabbit polyclonal antibodies against 13 proteins in the pheromone MAPK pathway.

Quantifying small numbers of antibodies with a 'near-universal' protein-DNA chimera.

We present general means to greatly increase the sensitivity of antibody-based assays. Augmentation relies on a 'tadpole' protein-DNA chimera whose protein moiety binds most classes of mammalian antibodies but not avian immunoglobulin Y (IgY). We used this tadpole in affinity capture assays followed by real-time PCR to quantify numerous molecules, including prostate-specific antigen (PSA) in human serum, with great sensitivity and accuracy.

Regulated cell-to-cell variation in a cell-fate decision system.

Here we studied the quantitative behaviour and cell-to-cell variability of a prototypical eukaryotic cell-fate decision system, the mating pheromone response pathway in yeast. We dissected and measured sources of variation in system output, analysing thousands of individual, genetically identical cells. Only a small proportion of total cell-to-cell variation is caused by random fluctuations in gene transcription and translation during the response ('expression noise').

A mutation in GerE that affects cotC promoter activation in Bacillus subtilis.

The DNA-binding protein GerE acts as both a repressor and an activator of transcription of genes transcribed by sigma(K)-RNA polymerase (RNA-P) during the later stages of endospore formation in Bacillus subtilis. GerE represses transcription from the sigK promoter, and activates transcription from other promoters, including cotC and cotX. Two different regions of GerE (AR1 and AR2) are required for activation of cotC and cotX, respectively.