Cliff I. Stains

Chemoselective Alteration of Fluorophore Scaffolds as a Strategy for the Development of Ratiometric Chemodosimeters

Ratiometric sensors generally couple binding events or chemical reactions at a distal site to changes in the fluorescence of a core fluorophore scaffold. However, such approaches are often hindered by spectral overlap of the product and reactant species. We provide a strategy to design ratiometric sensors that display dramatic spectral shifts by leveraging the chemoselective reactivity of novel functional groups inserted within fluorophore scaffolds. As a proof-of-principle, fluorophores containing a borinate (RF620 ) or silanediol (SiOH2R) functionality at the bridging position of the xanthene ring system are developed as endogenous H2 O2 sensors. Both these fluorophores display far-red to near-infrared excitation and emission prior to reaction. Upon oxidation by H2 O2 both sensors are chemically converted to tetramethylrhodamine, producing significant (≥66 nm) blue-shifts in excitation and emission maxima. This work provides a new concept for the development of ratiometric probes.

Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox-Based Sensors

Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation‐sensitive amino acid, termed CSox, to afford kinase‐selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox. Probes can be designed for either serine/threonine or tyrosine kinases, and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation, and application of CSox‐based protein kinase activity sensors. Curr. Protoc. Chem. Biol. 6:135‐156

An improved miniprotein host for fluorogenic supramolecular assembly on the surface of living cells

An improved miniprotein host capable of fluorogenic supramolecular assembly with a sI-Pht guest is reported. This improved host, termed 6.2.22, displays significant enhancements in both the EC50 for complexation and fluorescence activation of the sI-Pht guest, allowing for improved resolution of supramolecular complexation on the surface of living yeast.

Interrogating Protein Phosphatases with Chemical Activity Probes

Protein phosphatases, while long overlooked, have recently become appreciated as drivers of both normal- and disease-associated signaling events. As a result, the spotlight is now turning torwards this enzyme family and efforts geared towards the development of modern chemical tools for studying these enzymes are well underway. This Minireview focuses on the evolution of chemical activity probes, both optical and covalent, for the study of protein phosphatases. Small-molecule probes, global monitoring of phosphatase activity through the use of covalent modifiers, and targeted fluorescence-based activity probes are discussed. We conclude with an overview of open questions in the field and highlight the potential impact of chemical tools for studying protein phosphatases.

Identification of a fragmented small GTPase capable of conditional effector binding

A fragmented small GTPase capable of conditional effector binding is described. The effector binding function of this split-GTPase can be modulated using a small molecule input, thus allowing for the potential design of cellular signaling pathways.