Characterization of an N-Allylglyoxylamide-Based Bioorthogonal Nitrone Trap.

Aldehydes are attractive bioorthogonal coupling partners. The ease of manipulation of aldehydes and their orthogonality to other classes of bioorthogonal reactions have inspired the exploration of chemistries, which generate irreversible conjugates. Similarly, nitrones have been shown to be potent 1,3-dipoles in bioorthogonal reactions when paired with strained alkynes. Here, we combine the reactivity of nitrones with the simplicity of aldehydes using an N-allylglyoxylamide, in a cascade reaction with an N-alkylhydroxylamine to produce a bicyclic isoxazolidine. The reaction is found to be catalyzed by 5-methoxyanthranilic acid and proceeds at pH 7 with favorable kinetics. Using the HaloTag7 protein bearing an N-alkylhydroxylamine, we show the reaction to be bioorthogonal in a complex cell lysate and to proceed well at the surface of a HEK293 cell. Furthermore, the reaction is compatible with a typical strain-promoted alkyne-azide click reaction. The characteristics of this reaction suggest it will be a useful addition to the pallet of bioorthogonal reactions that have revolutionized chemical biology.

Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s.

Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

Point (S-to-G) Mutations in the W(S/G)GE Motif in Red/Green Cyanobacteriochrome GAF Domains Enhance Thermal Reversion Rates.

Cyanobacteriochromes (CBCRs) are photoreceptors consisting of single or tandem GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domains that bind bilin chromophores. Canonical red/green CBCR GAF domains are a well-characterized subgroup of the expanded red/green CBCR GAF domain family that binds phycocyanobilin (PCB) and converts between a thermally stable red-absorbing Pr state and a green-absorbing Pg state. The rate of thermal reversion from Pg to Pr varies widely among canonical red/green CBCR GAF domains, with half-lives ranging from days to seconds. Since the thermal reversion rate is an important parameter for the application of CBCR GAF domains as optogenetic tools, the molecular factors controlling the thermal reversion rate are of particular interest. Here, we report that point mutations in a well-conserved W(S/G)GE motif alter reversion rates in canonical red/green CBCR GAF domains in a predictable manner. Specifically, S-to-G mutations enhance thermal reversion rates, while the reverse, G-to-S mutations slow thermal reversion. Despite the distance (>10 Å) of the mutation site from the chromophore, molecular dynamics simulations and nuclear magnetic resonance (NMR) analyses suggest that the presence of a glycine residue allows the formation of a water bridge that alters the conformational dynamics of chromophore-interacting residues, leading to enhanced Pg to Pr thermal reversion.

Directed evolution approaches for optogenetic tool development.

Photoswitchable proteins enable specific molecular events occurring in complex biological settings to be probed in a rapid and reversible fashion. Recent progress in the development of photoswitchable proteins as components of optogenetic tools has been greatly facilitated by directed evolution approaches in vitro, in bacteria, or in yeast. We review these developments and suggest future directions for this rapidly advancing field.

Synergic Effects in the Activation of the Sweet Receptor GPCR Heterodimer for Various Sweeteners Predicted Using Molecular Metadynamics Simulations.

The sweet taste is elicited by activation of the TAS1R2/1R3 heterodimer G protein-coupled receptor. This is a therapeutic target for treatment of obesity and metabolic dysfunctions. Sweetener blends provide attractive strategies to lower the sugar level while preserving the attractive taste of food. To understand the synergic effect of various sweetener blend combinations of artificial and natural sweeteners, we carried out our molecular dynamics studies using predicted structures of the TAS1R2/1R3 heterodimer and predicted structures for the sweeteners. We used as a measure of activation the intracellular ionic lock distance between transmembrane helices 3 and 6 of TAS1R3. We find that full synergic combinations [rebaudioside A (Reb-A)/acesulfame K and Reb-A/sucralose] and partial synergic combinations (sucralose/acesulfame K) show significantly more negative changes in the free energy compared to single-ligand cases, while a pair known to be suppressive (saccharin and acesulfame K) shows significantly less changes than for the single-ligand case. This study provides an atomistic understanding of the mechanism for synergy and identifies new combinations of sweeteners to reduce the caloric content for treating diseases.