Monika G. Wood

HaloTag: a novel protein labeling technology for cell imaging and protein analysis.

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.

Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

Target engagement and drug residence time can be observed in living cells with BRET

The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment.

A luminescent biosensor with increased dynamic range for intracellular cAMP.

The second messenger cAMP is a key mediator of signal transduction following activation of G-protein coupled receptors. Investigations on Gs-coupled receptors would benefit from a second messenger assay that allows continuous monitoring of kinetic changes in cAMP concentration over a broad dynamic range. To accomplish this, we have evolved a luminescent biosensor for cAMP to better encompass the physiological concentration ranges present in living cells. When compared to an immunoassay, the evolved biosensor construct was able to accurately track both the magnitude and kinetics of cAMP change using a far less labor intensive format. We demonstrate the utility of this construct to detect a broad range of receptor activity, together with showing suitability for use in high-throughput screening.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

Darwinian natural selection for orange bioluminescent color in a Jamaican click beetle.

The Jamaican click beetle Pyrophorus plagiophthalamus (Coleoptera: Elateridae) is unique among all bioluminescent organisms in displaying a striking light color polymorphism [Biggley, W. H., Lloyd, J. E. & Seliger, H. H. (1967) J. Gen. Physiol. 50, 1681–1692]. Beetles on the island vary in the color of their ventral light organs from yellow–green to orange and their dorsal organs from green to yellow–green. The genetic basis for the color variation involves specific amino acid substitutions in the enzyme luciferase. Here, we show that dorsal and ventral light color in P. plagiophthalamus are under separate genetic control, we resolve the allelic basis for color variation, and, through analyses of luciferase sequence variation, we demonstrate that natural selection has produced a long-term adaptive trend for longer wavelength (more orange) ventral light on Jamaica. Our results constitute a novel example connecting the selective fixation of specific nucleotides in nature to their precisely determined phenotypic effects. We also present evidence suggesting that a recently derived ventral orange luciferase allele on the island has deterministically increased in frequency. Thus, the current luciferase polymorphism for P. plagiophthalamus appears to be mirroring the long-term anagenic trend on Jamaica, revealing a possible ongoing adaptive color transition in progress.

Bioluminescent assays for ADME evaluation: dialing in CYP selectivity with luminogenic substrates

Introduction: The cytochrome P450s (CYPs) are central to ADME studies because of their central role in drug metabolism. Proper CYP assay design and a correct understanding of CYP assay selectivity are critical for generating and interpreting biologically relevant data during drug development. Bioluminescent CYP assays use luminogenic probe substrates that have the unique property of producing photons in a second reaction with luciferase. Areas covered: This article presents the general design principles for in vitro CYP assays. Specifically, the article focuses on the bioluminescent approach that couples CYP activity with photon production. Expert opinion: Highly selective luminogenic substrates for CYP1A1, CYP1A2, CYP2C9, CYP3A4, CYP3A7, CYP4A and CYP4F have been developed with utility for interrogating the roles of these enzymes in biochemical and cell-based formats. These selective substrates are part of a larger collection of probes that deliver CYP inhibition and induction data that predict in vivo drug interactions. Furthermore, they support highly sensitive, rapid and scalable assays for cell-based and cell-free biochemical applications, which offer an alternative and often enabling option over conventional assay strategies.

Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system’s peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.Red-shifted bioluminescence emission is needed to improve deep tissue imaging resolution. Here, the authors develop a click beetle red luciferase mutant and two naphthyl-luciferin substrates, and show the ability of the new luciferin/luciferase pairing for deep tissue multispectral tomography in mice.