Synthetic auxotrophy in which bacterial
viability depends on the
presence of a synthetic amino acid provides a robust strategy for
the containment of genetically modified organisms and the development
of safe, live vaccines. However, a simple, general strategy to evolve
essential proteins to be dependent on synthetic amino acids is lacking.
Using a temperature-sensitive selection system, we evolved an Escherichia coli (E. coli) sliding clamp
variant with an orthogonal protein–protein interface, which
contains a Leu273 to p-benzoylphenyl alanine (pBzF)
mutation. The E. coli strain with this variant DNA
clamp has a very low escape frequency (<10–10), and its growth is strictly dependent on the presence of pBzF.
This selection strategy can be generally applied to create ncAA dependence
of other organisms with DNA clamp homologues.
The non-canonical amino acid l-(7-hydroxycoumarin-4-yl)ethylglycine can serve as a FRET acceptor from tryptophan. Here, we demonstrate how this amino acid pair can be used to generate an intrinsic FRET-based sensor of protein–ligand interactions.
Structural characterization of small molecules is a crucial component of organic synthesis. In this work, we applied microcrystal electron diffraction (MicroED) to analyze the structure of the product of an enzymatic reaction that was intended to produce the unnatural amino acid 2,4-dihydroxyphenylalanine (24DHF). Characterization of our isolated product with nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) suggested that an isomer of 24DHF had been formed. Microcrystals present in the isolated product were then used to determine its structure to 0.62 Å resolution, which confirmed its identity as 2-amino-2-(2,4-dihydroxyphenyl)propanoic acid (24DHPA). Moreover, the MicroED structural model indicated that both enantiomeric forms of 24DHPA were present in the asymmetric unit. Notably, the entire structure determination process including setup, data collection, and refinement was completed in ~1 h. The MicroED data not only bolstered previous results obtained using NMR and MS but also immediately provided information about the stereoisomers present in the product, which is difficult to achieve using NMR and MS alone. Our results therefore demonstrate that MicroED methods can provide useful structural information on timescales that are similar to many commonly used analytical methods and can be added to the existing suite of small molecule structure determination tools in future studies.
Fluorescent
noncanonical amino acids (fNCAAs) could serve as starting
points for the rational design of protein-based fluorescent sensors
of biological activity. However, efforts toward this goal are likely
hampered by a lack of atomic-level characterization of fNCAAs within
proteins. Here, we describe the spectroscopic and structural characterization
of five streptavidin mutants that contain the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine
(7-HCAA) at sites proximal to the binding site of its substrate, biotin.
Many of the mutants exhibited altered fluorescence spectra in response
to biotin binding, which included both increases and decreases in
fluorescence intensity as well as red- or blue-shifted emission maxima.
Structural data were also obtained for three of the five mutants.
The crystal structures shed light on interactions between 7-HCAA and
functional groups, contributed either by the protein or by the substrate,
that may be responsible for the observed changes in the 7-HCAA spectra.
These data could be used in future studies aimed at the rational design
of fluorescent, protein-based sensors of small molecule binding or
dissociation.
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