The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc.

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.

The Expresso® Solubility and Expression Screening System uses a simple ligase-free cloning strategy and a single PCR product to construct and test seven distinct protein fusion partners in parallel over the course of a few days. This multiplexed system simplifies the screening of fusion partners and greatly increases the chances of obtaining improved expression and solubility of target proteins.

Use of fluorescent proteins to study in vivo processes in mammalian systems begs development of near-infrared (NIR) biomarkers due to superior penetration of the excitation light at longer wavelengths. Bacteriophytochromes (BphPs) that use biliverdin as their chromophore have been engineered to form monomeric NIR biomarkers. Absorption of a red light photon (l=700 nm) leads to isomerization of the C15=C16 bond of BV in the Pr ground state, leading to the second ground state, the far red-light absorbing Pfr. The archetypal NIR phytofluor carried a D207H substitution. The 207 position is important because the main chain carbonyl forms a hydrogen bond with the BV A ring, thus potentially functioning as a proton sink during the photocycle. This variant spurred development of NIR biomarker BPhPs IFP1.4, Wi-Phy, and iRFP. We have solved the structure of IFP1.4 and observe a tightly packed hydrophobic core in the BV binding pocket in contrast to the open pocket in native BphP. Side chains of V173, M174, and V288 form this stabilizing interaction, which is absent in the wild type counterpart because residue 288 is an alanine. We have further disproven the hypothesis that H207 is required for fluorescence, by engineering IFP1.4 D207 and demonstrating it is brighter than any NIR phytofluor described to date. Our research has helped delineate the origin of fluorescence in BphPs and will help phytofluors gain wide spread use as their brightness and resistance to photobleaching are augmented.

Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) biomarkers that exploit the ability of light in this range to penetrate tissue. Bacteriophytochromes (BphPs) are photoreceptors that couple absorbance of NIR light to photoisomerization, protein conformational changes, and signal transduction. BphPs have been engineered to form NIR fluorophores, including IFP1.4, Wi-Phy, and the iRFP series, initially by replacement of Asp-207 by His. This position was suggestive because its main chain carbonyl is within hydrogen-bonding distance to pyrrole ring nitrogens of the biliverdin chromophore, thus potentially functioning as a crucial transient proton sink during photoconversion. To explain the origin of fluorescence in these phytofluors, we solved the crystal structures of IFP1.4 and a comparison non-fluorescent monomeric phytochrome DrCBD(mon). Met-186 and Val-288 in IFP1.4 are responsible for the formation of a tightly packed hydrophobic hub around the biliverdin D ring. Met-186 is also largely responsible for the blue-shifted IFP1.4 excitation maximum relative to the parent BphP. The structure of IFP1.4 revealed decreased structural heterogeneity and a contraction of two surface regions as direct consequences of side chain substitutions. Unexpectedly, IFP1.4 with Asp-207 reinstalled (IFPrev) has a higher fluorescence quantum yield (approximate to 9%) than most NIR phytofluors published to date. In agreement, fluorescence lifetime measurements confirm the exceptionally long excited state lifetimes, up to 815 ps, in IFP1.4 and IFPrev. Our research helps delineate the origin of fluorescence in engineered BphPs and will facilitate the wide-spread adoption of phytofluors as biomarkers.

Specialized methylketone-containing metabolites accumulate in certain plants, in particular wild tomatoes in which they serve as toxic compounds against chewing insects. In Solanum habrochaites f. glabratum, methylketone biosynthesis occurs in the plastids of glandular trichomes and begins with intermediates of de novo fatty acid synthesis. These fatty-acyl intermediates are converted via sequential reactions catalyzed by Methylketone Synthase2 (MKS2) and MKS1 to produce the n-1 methylketone. We report crystal structures of S. habrochaites MKS1, an atypical member of the α/β-hydrolase superfamily. Sequence comparisons revealed the MKS1 catalytic triad, Ala-His-Asn, as divergent to the traditional α/β-hydrolase triad, Ser-His-Asp. Determination of the MKS1 structure points to a novel enzymatic mechanism dependent upon residues Thr-18 and His-243, confirmed by biochemical assays. Structural analysis further reveals a tunnel leading from the active site consisting mostly of hydrophobic residues, an environment well suited for fatty-acyl chain binding. We confirmed the importance of this substrate binding mode by substituting several amino acids leading to an alteration in the acyl-chain length preference of MKS1. Furthermore, we employ structure-guided mutagenesis and functional assays to demonstrate that MKS1, unlike enzymes from this hydrolase superfamily, is not an efficient hydrolase but instead catalyzes the decarboxylation of 3-keto acids.

Phytochrome is a multidomain dimeric red light photoreceptor that utilizes a chromophore-binding domain (CBD), a PHY domain, and an output module to induce cellular changes in response to light. A promising biotechnology tool emerged when a structure-based substitution at Asp-207 was shown to be an infrared fluorophore that uses a biologically available tetrapyrrole chromophore. We report multiple crystal structures of this D207H variant of the Deinococcus radiodurans CBD, in which His-207 is observed to form a hydrogen bond with either the tetrapyrrole A-ring oxygen or the Tyr-263 hydroxyl. Based on the implications of this duality for fluorescence properties, Y263F was introduced and shown to have stronger fluorescence than the original D207H template. Our structures are consistent with the model that the Y263F change prevents a red light-induced far-red light absorbing phytochrome chromophore configuration. With the goal of decreasing size and thereby facilitating use as a fluorescent tag in vivo, we also engineered a monomeric form of the CBD. Unexpectedly, photoconversion was observed in the monomer despite the lack of a PHY domain. This observation underscores an interplay between dimerization and the photochemical properties of phytochrome and suggests that the monomeric CBD could be used for further studies of the photocycle. The D207H substitution on its own in the monomer did not result in fluorescence, whereas Y263F did. Combined, the D207H and Y263F substitutions in the monomeric CBD lead to the brightest of our variants, designated Wisconsin infrared phytofluor (Wi-Phy).

Phytochromes are environmental sensors, historically thought of as red/far-red photoreceptors in plants. Their photoperception occurs through a covalently linked tetrapyrrole chromophore, which undergoes a light-dependent conformational change propagated through the protein to a variable output domain. The phytochrome composition is modular, typically consisting of a PAS-GAF-PHY architecture for the N-terminal photosensory core. A collection of three-dimensional structures has uncovered key features, including an unusual figure-of-eight knot, an extension reaching from the PHY domain to the chromophore-binding GAF domain, and a centrally located, long α-helix hypothesized to be crucial for intramolecular signaling. Continuing identification of phytochromes in microbial systems has expanded the assigned sensory abilities of this family out of the red and into the yellow, green, blue, and violet portions of the spectrum. Furthermore, phytochromes acting not as photoreceptors but as redox sensors have been recognized. In addition, architectures other than PAS-GAF-PHY are known, thus revealing phytochromes to be a varied group of sensory receptors evolved to utilize their modular design to perceive a signal and respond accordingly. This review focuses on the structures of bacterial phytochromes and implications for signal transmission. We also discuss the small but growing set of bacterial phytochromes for which a physiological function has been ascertained.

The trichomes of the wild tomato species Solanum habrochaites subsp. glabratum synthesize and store high levels of methylketones, primarily 2-tridecanone and 2-undecanone, that protect the plants against various herbivorous insects. Previously, we identified cDNAs encoding two proteins necessary for methylketone biosynthesis, designated methylketone synthase 1 (ShMKS1) and ShMKS2. Here, we report the isolation of genomic sequences encoding ShMKS1 and ShMKS2 as well as the homologous genes from the cultivated tomato, Solanum lycopersicum. We show that a full-length transcript of ShMKS2 encodes a protein that is localized in the plastids. By expressing ShMKS1 and ShMKS2 in Escherichia coli and analyzing the products formed, as well as by performing in vitro assays with both ShMKS1and ShMKS2, we conclude that ShMKS2 acts as a thioesterase hydrolyzing 3-ketoacyl-acyl carrier proteins (plastid-localized intermediates of fatty acid biosynthesis) to release 3-ketoacids and that ShMKS1 subsequently catalyzes the decarboxylation of these liberated 3-ketoacids, forming the methylketone products. Genes encoding proteins with high similarity to ShMKS2, a member of the “hot-dog fold” protein family that is known to include other thioesterases in nonplant organisms, are present in plant species outside the genus Solanum. We show that a related enzyme from Arabidopsis (Arabidopsis thaliana) also produces 3-ketoacids when recombinantly expressed in E. coli. Thus, the thioesterase activity of proteins in this family appears to be ancient. In contrast, the 3-ketoacid decarboxylase activity of ShMKS1, which belongs to the α/β-hydrolase fold superfamily, appears to have emerged more recently, possibly within the genus Solanum.

The oxidative cleavage of carotenoids leads to the production of apocarotenoids and is catalyzed by a family of carotenoid cleavage dioxygenases (CCDs). CCDs often exhibit substrate promiscuity, which probably contributes to the diversity of apocarotenoids found in nature. Biologically and commercially important apocarotenoids include the phytohormone abscisic acid, the visual and signaling molecules retinal and retinoic acid, and the aromatic volatile beta-ionone. Unexpected properties associated with the CCD catalytic products emphasize their role in many aspects of plant growth and development. For instance, CCD7 and CCD8 produce a novel, graft-transmissible hormone that controls axillary shoot growth in plants. Here, CCDs are discussed according to their roles in the biosynthesis of these products. Recent studies regarding their mechanism of action are also addressed.