Mechanistic Basis for Red Light Switching of Azonium Ions.

Azonium ions formed by the protonation of tetra--methoxy-substituted aminoazobenzenes photoisomerize with red light under physiological conditions. This property makes them attractive as molecular tools for the photocontrol of physiological processes, for example, in photopharmacology. However, a mechanistic understanding of the photoisomerization process and subsequent thermal relaxation is necessary for the rational application of these compounds as well as for guiding the design of derivatives with improved properties.

Isomerization of bioactive acylhydrazones triggered by light or thiols.

The acylhydrazone unit is well represented in screening databases used to find ligands for biological targets, and numerous bioactive acylhydrazones have been reported. However, potential E/Z isomerization of the C=N bond in these compounds is rarely examined when bioactivity is assayed. Here we analysed two ortho-hydroxylated acylhydrazones discovered in a virtual drug screen for modulators of N-methyl-D-aspartate receptors and other bioactive hydroxylated acylhydrazones with structurally defined targets reported in the Protein Data Bank.

Synthesis and Optimization of Collagen-targeting Peptide-Glycosaminoglycans for Inhibition of Platelets Following Endothelial Injury.

Many endothelial complications, whether from surgical or pathological origins, can result in the denudation of the endothelial layer and the exposure of collagen. Exposure of collagen results in the activation of platelets, leading to thrombotic and inflammatory cascades that ultimately result in vessel stenosis. We have previously reported the use of peptide-GAG compounds to target exposed collagen following endothelial injury.

Modification of proteins with azobenzene crosslinkers using reversible covalent bonds.

Thiol-reactive reagents designed for the chemical modification of proteins cannot, in general, be used directly for the modification of intracellular targets because the presence of millimolar concentrations of glutathione inside cells effectively outcompetes reaction with target thiols. Here we report an equilibrium, entropic strategy for achieving target selectivity using a cyanoacrylate-based thiol-reactive cross-linker (BCNA) with two reactive sites. This compound exhibits ≳200-fold selectivity for reaction with target peptides and proteins containing appropriately spaced pairs of thiols, reaction with mono-thiols.

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.

An anti-PD-1-GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy.

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies.

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.

Structure-based design of a photoswitchable affibody scaffold.

Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding.

Localized inhibition of platelets and platelet derived growth factor by a matrix targeted glycan mimetic significantly attenuates liver fibrosis.

New therapeutic strategies are needed for the growing unmet clinical needs in liver disease and fibrosis. Platelet activation and PDGF activity are recognized as important therapeutic targets; however, no therapeutic approach has yet addressed these two upstream drivers of liver fibrosis. We therefore designed a matrix-targeting glycan therapeutic, SBR-294, to inhibit collagen-mediated platelet activation while also inhibiting PDGF activity.

Yeast Two-Hybrid Screening of Photoswitchable Protein-Protein Interaction Libraries.

Although widely used in the detection and characterization of protein-protein interactions, Y2H screening has been under-used for the engineering of new optogenetic tools or the improvement of existing tools. Here we explore the feasibility of using Y2H selection and screening to evaluate libraries of photoswitchable protein-protein interactions. We targeted the interaction between circularly permuted photoactive yellow protein (cPYP) and its binding partner binder of PYP dark-state (BoPD) by mutating a set of four surface residues of cPYP that contribute to the binding interface.

Synthesis and characterization of bis(4-amino-2-bromo-6-methoxy)azobenzene derivatives.

Aminoazobenzene derivatives with four substituents with respect to the N-N double bond are a relatively unexplored class of azo compounds that show promise for use as photoswitches in biology. Tetra--methoxy-substituted aminoazobenzene compounds in particular can form azonium ions under physiological conditions and exhibit red-light photoswitching. Here, we report the synthesis and characterization of two bis(4-amino-2-bromo-6-methoxy)azobenzene derivatives.

A time-dependent role for the transcription factor CREB in neuronal allocation to an engram underlying a fear memory revealed using a novel in vivo optogenetic tool to modulate CREB function.

The internal representation of an experience is thought to be encoded by long-lasting physical changes to the brain ("engrams") . Previously, we and others showed within the lateral amygdala (LA), a region critical for auditory conditioned fear, eligible neurons compete against one other for allocation to an engram. Neurons with relatively higher function of the transcription factor CREB were more likely to be allocated to the engram.

Intramolecular cross-linking of proteins with azobenzene-based cross-linkers.

The photo-control of protein activity can often be achieved via the photo-control of protein structure. Intramolecular cross-linkers that change length upon photoisomerization provide a means to photo-control protein structure by linking to pairs of Cys residues in a protein sequence. In this protocol, we describe general methods for introducing intramolecular cross-linkers, both UV light switchable and red-light switchable, under either denaturing or native conditions.

Photoswitchable affinity reagents: Computational design and efficient red-light switching.

Photo-controlled affinity reagents seek to provide modular spatiotemporal control of bioactivity by conferring photo-switchability of function on an affinity reagent scaffold. Here we used Rosetta-based computational methods to screen for sites on the Fynomer affinity reagent structure for attachment of photoswitchable cross-linkers. Both established UV-based cross-linkers (azobenzene-iodoacetamide (IAC)) and an azonium-based efficient red light switchable cross-linker, piperazino-tetra--methoxy azobenzene (PIP), were then tested experimentally.

Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.

We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions.

A Yeast System for Discovering Optogenetic Inhibitors of Eukaryotic Translation Initiation.

The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development, and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis noninvasively within minutes and with a spatial scale as small as a single synapse.

Selection of Protein-Protein Interactions of Desired Affinities with a Bandpass Circuit.

We have developed a genetic circuit in Escherichia coli that can be used to select for protein-protein interactions of different strengths by changing antibiotic concentrations in the media. The genetic circuit links protein-protein interaction strength to β-lactamase activity while simultaneously imposing tuneable positive and negative selection pressure for β-lactamase activity. Cells only survive if they express interacting proteins with affinities that fall within set high- and low-pass thresholds; i.

Discovering Selective Binders for Photoswitchable Proteins Using Phage Display.

Nature provides an array of proteins that change conformation in response to light. The discovery of a complementary array of proteins that bind only the light-state or dark-state conformation of their photoactive partner proteins would allow each light-switchable protein to be used as an optogenetic tool to control protein-protein interactions. However, as many photoactive proteins have no known binding partner, the advantages of optogenetic control-precise spatial and temporal resolution-are currently restricted to a few well-defined natural systems.

Duplication of a Single Strand in a β-Sheet Can Produce a New Switching Function in a Photosensory Protein.

Duplication of a single β-strand that forms part of a β-sheet in photoactive yellow protein (PYP) was found to produce two approximately isoenergetic protein conformations, in which either the first or the second copy of the duplicated β-strand participates in the β-sheet. Whereas one conformation (big-loop) is more stable at equilibrium in the dark, the other conformation (long-tail) is populated after recovery from blue light irradiation. By appending a recognition motif (E-helix) to the C-terminus of the protein, we show that β-strand duplication, and the resulting possibility of β-strand slippage, can lead to a new switchable protein-protein interaction.

PrismPlus: a mouse line expressing distinct fluorophores in four different brain cell types.

To screen the complex central nervous system (CNS) injury responses, we created a quadruple-labelled 'PrismPlus' mouse line with a genetically encoded distinct fluorescent tag in oligodendrocytes, microglia, neurons, and astrocytes. Cx3cr1-gfp and Prism mice originally developed by Jung et al., 2000 and Dougherty et al.

Computational Design of a Photocontrolled Cytosine Deaminase.

There is growing interest in designing spatiotemporal control over enzyme activities using noninvasive stimuli such as light. Here, we describe a structure-based, computation-guided predictive method for reversibly controlling enzyme activity using covalently attached photoresponsive azobenzene groups. Applying the method to the therapeutically useful enzyme yeast cytosine deaminase, we obtained a ∼3-fold change in enzyme activity by the photocontrolled modulation of the enzyme's active site lid structure, while fully maintaining thermostability.

Near-Infrared Photoswitching of Azobenzenes under Physiological Conditions.

Biological tissue exhibits an absorbance minimum in the near-infrared between 700 and 900 nm that permits deep penetration of light. Molecules that undergo photoisomerization in this bio-optical window are highly desirable as core structures for the development of photopharmaceuticals and as components of chemical-biological tools. We report the systematic design, synthesis, and testing of an azobenzene derivative tailored to undergo single-photon photoswitching with near-infrared light under physiological conditions.

Strategies for the photo-control of endogenous protein activity.

Photo-controlled or 'optogenetic' effectors interfacing with endogenous protein machinery allow the roles of endogenous proteins to be probed. There are two main approaches being used to develop optogenetic effectors: (i) caging strategies using photo-controlled conformational changes, and (ii) protein relocalization strategies using photo-controlled protein-protein interactions. Numerous specific examples of these approaches have been reported and efforts to develop general methods for photo-control of endogenous proteins are a current focus.