Bottom-Up Design: A Modular Golden Gate Assembly Platform of Yeast Plasmids for Simultaneous Secretion and Surface Display of Distinct FAP Fusion Proteins.

A need in synthetic biology is the ability to precisely and efficiently make flexible fully designed vectors that addresses challenging cloning strategies of single plasmids that rely on combinatorial co-expression of a multitude of target and bait fusion reporters useful in projects like library screens. For these strategies, the regulatory elements and functional components need to correspond perfectly to project specific sequence elements that facilitate easy exchange of these elements. This requires systematic implementation and building on recent improvements in Golden Gate (GG) that ensures high cloning efficiency for such complex vectors. Currently, this is not addressed in the variety of molecular GG cloning techniques in synthetic biology. Here, we present the bottom-up design and plasmid synthesis to prepare 10 kb functional yeast secrete and display plasmids that uses an optimized version of GG in combination with fluorogen-activating protein reporter technology. This allowed us to demonstrate nanobody/target protein interactions in a single cell, as detected by cell surface retention of secreted target proteins by cognate nanobodies. This validates the GG constructional approach and suggests a new approach for discovering protein interactions. Our GG assembly platform paves the way for vector-based library screening and can be used for other recombinant GG platforms.

Activity-evoked and spontaneous opening of synaptic fusion pores.

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. Here, synaptic DCV fusion pore openings are imaged without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca2+ SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission.

Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inward rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorogen-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast.

Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.

A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules.

Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields.

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications.

Engineering fluorogen activating proteins into self-assembling materials.

We present herein characteristics of a conjugate in which dL5, a fluorogen activating protein (FAP), and AEAEAKAK, an amphiphilic peptide, are combined to form a solid-phase fluorescence detection platform. The FAP dL5 is a covalently linked dimer of two identical light chain variable fragments which activates the fluorescence of the fluorogen malachite green (MG). The amphiphilic peptide of sequence AEAEAKAK is a building block of stimuli-responsive materials that undergoes sol-gel phase transition at high ionic strengths. We hypothesize that the novel bifunctional protein containing both the FAP and the amphiphile, termed dL5_EAK coassembles with the self-assembling peptide [AEAEAKAK]2 (EAK16-II) to form an insoluble membrane composite whereby the fluorescence enhancement function of the FAP domain remains intact. Denaturing polyacrylamide electrophoresis indicated that greater than 78% of dL5_EAK incorporates into the EAK16-II membrane. Conversely, less than 32% of dL5 without the EAK sequence associates with the insoluble fraction of EAK16-II in buffers. Membranes containing dL5_EAK and EAK16-II exhibited at least 4-fold higher fluorescence intensity compared to mixtures containing dL5 and EAK16-II. Scanning electron microscopy revealed the presence of particulates, presumably FAPs, scattered on the membrane fibrils. The evidence suggests a system of materials that can be developed into in situ forming local sensors by immobilizing dL5 into coacervate, on which MG can be detected. It is envisioned that dL5 membranes can be established in diseased locales to monitor infiltration and migration of inflammatory cells marked with antibodies conjugated to MG.

Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors.

The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g., EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes ('activate the fluorogen'). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (ß2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.

Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.

We have developed a class of dendron-based fluorogenic dyes (termed dyedrons) comprised of multiple cyanine (Cy3) donors coupled to a single malachite green (MG) acceptor that fluoresce only when the MG is noncovalently but specifically bound to a cognate single chain antibody (scFv). These cell-impermeant dyedrons exploit efficient intramolecular energy transfer from Cy3 donors to stoichiometrically amplify the fluorescence of MG chromophores that are activated by binding to the scFv. These chromophore enhancements, coupled with our optimized scFv, can significantly increase fluorescence emission generated by the dyedron/scFv complex to brightness levels several-fold greater than that for single fluorescent proteins and targeted small molecule fluorophores. Efficient intramolecular quenching of free dyedrons enables sensitive homogeneous (no wash) detection under typical tissue culture conditions, with undetectable nonspecific activation.

Fluorogen-activating single-chain antibodies for imaging cell surface proteins.

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.