Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

We report that the recently introduced commercial strain of V. natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 non-canonical L-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recoded E. coli strains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of mis-incorporated natural L-amino acids at the UAG-programmed position, especially in cases where the ncAA is an imperfect substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve specific activity of highly desirable but imperfect ncAA substrates.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis

Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh. In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2. Our data, combined with previous studies, provide the molecular sequence of events in the coupled binding of sodium and aspartate to GltPh.

Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the vertebrate nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type iGluRs using genetically encoded unnatural amino acid (UAA) photocross-linkers, p-benzoyl-l-phenylalanine (BzF) and p-azido-l-phenylalanine (AzF). We introduced these UAAs at sites throughout the TMD of the GluA2 receptor and characterized the mutants in patch-clamp recordings, exposing them to glutamate and ultraviolet (UV) light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence for an interaction between the Pre-M1 and the M4 TMD helix during desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter in the M2 segment, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric ion channels, selectivity filter gating.

Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of AMPA-type iGluRs using genetically-encoded unnatural amino acid (UAA) photo-crosslinkers, p-benzoyl-L-phenylalanine (BzF) and p-azido-L-phenylalanine (AzF). We introduced UAAs at sites throughout the TMD of the GluA2 receptor and characterized these mutants in patch-clamp recordings, exposing them to glutamate and UV light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence that an interaction between the Pre-M1 and the M4 TMD helix was essential for normal activation and desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric channels, selectivity filter gating.