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Summary
By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch-complex with a 200 nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems.
Sucralose
is a commonly employed artificial sweetener that behaves
very differently than its natural disaccharide counterpart, sucrose,
in terms of its interaction with biomolecules. The presence of sucralose
in solution is found to destabilize the native structure of two model
protein systems: the globular protein bovine serum albumin and an
enzyme staphylococcal nuclease. The melting temperature of these proteins
decreases as a linear function of sucralose concentration. We correlate
this destabilization to the increased polarity of the molecule. The
strongly polar nature is manifested as a large dielectric friction
exerted on the excited-state rotational diffusion of tryptophan using
time-resolved fluorescence anisotropy. Tryptophan exhibits rotational
diffusion proportional to the measured bulk viscosity for sucrose
solutions over a wide range of concentrations, consistent with a Stokes–Einstein
model. For sucralose solutions, however, the diffusion is dependent
on the concentration, strongly diverging from the viscosity predictions,
and results in heterogeneous rotational diffusion.
A major challenge in carbon‒hydrogen (C‒H) bond functionalization is to have the catalyst control precisely where a reaction takes place. In this study, we report engineered cytochrome P450 enzymes that perform unprecedented enantioselective C‒H amidation reactions and control the site selectivity to divergently construct β-, γ-, and δ-lactams, completely overruling the inherent reactivities of the C‒H bonds. The enzymes, expressed in Escherichia coli cells, accomplish this abiological carbon‒nitrogen bond formation via reactive iron-bound carbonyl nitrenes generated from nature-inspired acyl-protected hydroxamate precursors. This transformation is exceptionally efficient (up to 1,020,000 total turnovers) and selective (up to 25:1 regioselectivity and 97%, please refer to compound 2v enantiomeric excess), and can be performed easily on preparative scale.
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