HaloTag is a small self‐labeling protein that is frequently used for creating fluorescent reporters in living cells. The small‐molecule dyes used with HaloTag are almost exclusively based on rhodamine scaffolds, which are often expensive and challenging to synthesize. Herein, we report the engineering of HaloTag for use with a chemically accessible, inexpensive fluorophore based on the dimethylamino‐styrylpyridium dye. Through directed evolution, the maximum fluorogenicity and the apparent second‐order bioconjugation rate constants could be improved up to 4‐fold and 42‐fold, respectively. One of the top variants, HT‐SP5, enabled reliable imaging in mammalian cells, with a 113‐fold fluorescence enhancement over the parent protein. Additionally, crystallographic characterization of selected mutants suggests the chemical origin of the fluorescent enhancement. The improved dye system offers a valuable tool for imaging and illustrates the viability of engineering self‐labeling proteins for alternative fluorophores.

The selective functionalization of sp 3 C–H bonds is a versatile tool for the diversification of organic compounds. Combining attractive features of homogeneous and enzymatic catalysts, artificial metalloenzymes offer an ideal means to selectively modify these inert motifs. Herein, we report on a copper(I) heteroscorpionate complex embedded within streptavidin that catalyzes the intramolecular insertion of a carbene into sp 3 C–H bonds. Target residues for genetic optimization of the artificial metalloenzyme were identified by quantum mechanics/molecular mechanics simulations. Double-saturation mutagenesis yielded detailed insight on the contribution of individual amino acids on the activity and the selectivity of the artificial metalloenzyme. Mutagenesis at a third position afforded a set of artificial metalloenzymes that catalyze the enantio- and regioselective formation of β- and γ-lactams with high turnovers and promising enantioselectivities.

Artificial metalloenzymes result from anchoring a metal cofactor within a host protein. Such hybrid catalysts combine the selectivity and specificity of enzymes with the versatility of (abiotic) transition metals to catalyze new-to-nature reactions in an evolvable scaffold. With the aim of improving the localization of an arylsulfonamide-bearing iridium-pianostool catalyst within human carbonic anhydrase II (hCAII) for the enantioselective reduction of prochiral imines, we introduced a covalent linkage between the host and the guest. Herein, we show that a judiciously positioned cysteine residue reacts with a p-nitropicolinamide ligand bound to iridium to afford an additional sulfonamide covalent linkage. Three rounds of directed evolution, performed on the dually anchored cofactor, led to improved activity and selectivity for the enantioselective reduction of harmaline (up to 97% ee (R) and >350 turnovers on a preparative scale). To evaluate the substrate scope, the best hits of each generation were tested with eight substrates. X-ray analysis, carried out at various stages of the evolutionary trajectory, was used to scrutinize (i) the nature of the covalent linkage between the cofactor and the host as well as (ii) the remodeling of the substrate-binding pocket.

Background To evaluate the midterm follow-up and 5-year survival outcome of the minimally invasive direct coronary artery bypass (MIDCAB) procedure compared with the survival of the general Swiss population. Methods Retrospective study on preoperative data, intraoperative data, and postoperative outcome of patients who underwent MIDCAB surgery between June 2010 and February 2019. To assess validity of this surgical therapy, outcomes were compared with survival data of a gender- and age-matched cohort of the general Swiss population taken from the database of the Swiss Federal Statistical Office. Results A total of 88 patients were included. Median (interquartile range [IQR[) age was 66 (56–75) years, and 27% (n = 24) were female. The median (IQR) length of the in-hospital stay was 7 (6–8) days. No postoperative stroke occurred. The 30-day mortality was 1.1% (n = 1). Reintervention for failed left internal mammary artery was needed in 1.1% (n = 1). The median (IQR) ejection fraction was 58% (47–60) preoperatively and remained stable during follow-up. The median (IQR) follow-up period was 3 (1.1–5.2) years. Five years postoperatively, 83% (confidence interval, 69–91) of the patients were alive, showing an overlap with the range of survival of the matched subcohort of the general Swiss population (range, 84–100%). Conclusion Though suffering from coronary heart disease, patients after MIDCAB show almost equal survival rates as an equivalent subcohort corresponding to the general Swiss population matched on age and gender. Thus, our data show this treatment to be safe and beneficial.

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