Multiplexed no-wash cellular imaging using BenzoTag, an evolved self-labeling protein.

Self-labeling proteins are powerful tools for exploring biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye. HaloTag7 is the most popular self-labeling protein due to its broad utility, its bio-orthogonality, and the simplicity of its chloroalkane ligand. However, reaction rates of HaloTag7 with different chloroalkane-containing substrates are highly variable and rates are only very fast for rhodamine-based dyes.

BenzoHTag, a fluorogenic self-labeling protein developed using molecular evolution.

Self-labeling proteins are powerful tools in chemical biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye, with the genetic specificity of a protein fusion. HaloTag7 is the most popular self-labeling protein due to its fast labeling kinetics and the simplicity of its chloroalkane ligand. Reaction rates of HaloTag7 with different chloroalkane-containing substrates is highly variable and rates are only very fast for rhodamine-based dyes.

Engineered fluorogenic HaloTag ligands for turn-on labelling in live cells.

Recent years have seen dramatic improvements in the design of organic fluorophores based on limiting non-radiative decay pathways. We sought to extend this understanding to benzothiadiazoles that have been used as turn-on fluorescent substrates for the self-labeling protein HaloTag. When conjugated to HaloTag, the benzothiadiazoles reside in a narrow tunnel that precludes twisted internal charge transfer, which allowed us to explore steric and electronic effects on other non-radiative decay pathways.

Hydrogen Bond and Geometry Effects of Thioamide Backbone Modifications.

Thioamide substitution of backbone peptide bonds can probe interactions along the main chain of proteins. Despite theoretical predictions of the enhanced hydrogen bonding propensities of thioamides, previous studies often do not consider the geometric constraints imposed by folded peptide secondary structure. This work addresses drawbacks in previous studies that ignored the geometry dependence and local dielectric properties of thioamide hydrogen bonding and identifies cases where thioamides may be either stronger or weaker hydrogen-bonding partners than amides.

Stapled β-Hairpins Featuring 4-Mercaptoproline.

Peptides constrained by intramolecular cross-links, especially stapled α-helices, have emerged as versatile scaffolds for drug development. However, there are fewer examples of similarly constrained scaffolds for other secondary structures. Here, we used a novel computational strategy to identify an optimal staple for antiparallel β-strands, and then we incorporated that staple within a β-hairpin peptide.