Structural Origins of Altered Spectroscopic Properties upon Ligand Binding in Proteins Containing a Fluorescent Noncanonical Amino Acid.

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.

Heterologous expression and purification of the bicarbonate transporter BicA from Synechocystis sp. PCC 6803.

The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and protocols for purification are required. In this work we have optimized the expression and purification of BicA from strain Synechocystis sp. PCC 6803 (BicA6803) in Escherichia coli. It was determined that expression with C43 (DE3) Rosetta2 at 37 °C produced the highest levels of over-expressed BicA6803 relative to other strains screened, and membrane solubilization with n-dodecyl-ß-d-maltopyranoside facilitated the purification of high levels of stable and homogenous BicA6803. Using these expression and purification strategies, the final yields of purified BicA were 6.5 ± 1.0 mg per liter of culture.

An intrinsic FRET sensor of protein-ligand interactions.

We describe an approach for the development of fluorescent sensors of metabolite binding in which a genetically encoded fluorescent non-canonical amino acid (fNCAA) containing a 7-hydroxycoumarin moiety (7-HCAA) forms a FRET pair with native tryptophan residues. Although previous studies demonstrated the potential for using 7-HCAA as an acceptor for tryptophan, this approach has not yet been explored within a single protein containing multiple tryptophan residues. A structure-based analysis of a hexokinase enzyme with multiple native tryptophan residues identified glutamate 50 as a potential site of 7-HCAA incorporation; Glu50 moves closer to the native tryptophans upon substrate binding. Substitution of 7-HCAA at residue 50 led to an increase in FRET efficiency in the presence of the substrate; this effect was not observed in a control protein where no change in distance between 7-HCAA and the native tryptophans occurs on substrate binding. This system was then used to directly observe differences in binding affinity of the hexokinase that occur at a number of pH values. Our approach builds on previous research in that it eliminates the need for the incorporation of multiple fNCAAs or fluorescent labels within a target protein and can be used to study substrate binding with native ligands. As such, it serves to expand the versatility of FRET-based techniques.

A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth.

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.

Rapid Structural Analysis of a Synthetic Non-canonical Amino Acid by Microcrystal Electron Diffraction.

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.