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.

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.

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.

Improving a designed photocontrolled DNA-binding protein.

Photocontrolled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photocontrolled DNA-binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from Halorhodospira halophila [Morgan, S. A.

A collection of caged compounds for probing roles of local translation in neurobiology.

Spatially localized translation plays a vital role in the normal functioning of neuronal systems and is widely believed to be involved in both learning and memory formation. It is of central interest to understand both the phenomenon and molecular mechanisms of local translation using new tools and approaches. Caged compounds can, in principle, be used as tools to investigate local translation since optical activation of bioactive molecules can achieve both spatial and temporal resolution on the micron scale and on the order of seconds or less, respectively.

Reversible photocontrol of DNA binding by a designed GCN4-bZIP protein.

Synthetic photocontrolled proteins could be powerful tools for probing cellular chemistry. Several previous attempts to produce such systems by incorporating photoisomerizable chromophores into biomolecules have led to photocontrol but with incomplete reversibility, where the chromophore becomes trapped in one photoisomeric state. We report here the design of a modified GCN4-bZIP DNA-binding protein with an azobenzene chromophore introduced between Cys residues at positions 262 and 269 (S262C, N269C) within the zipper domain.

Site-specific biosynthetic incorporation of a fluorescent tag into proteins via cysteine-tRNA(Cys).

Site-specific incorporation of biophysical probes into proteins during translation can permit structure/function studies on selected proteins in heterogeneous environments. We report here a procedure for incorporating a fluorescent tag into proteins via Escherichia coli Cys-tRNA(Cys) during in vitro protein synthesis. Naturally occurring Cys-tRNA(Cys) is an attractive vehicle for fluorophore incorporation since it can be readily prepared in quantity and reacted with commercially available fluorophores.