Fluorinated Tags to Study Protein Conformation and Interactions Using 19F NMR.

The incorporation of fluorine atoms into a biomacromolecule provides a background-free and environmentally sensitive reporter of structure, conformation and interactions using 19F NMR. There are several methods to introduce the 19F reporter - either by synthetic incorporation via solid phase peptide synthesis; by suppressing the incorporation or biosynthesis of a natural amino acid and supplementing the growth media with a fluorinated counterpart during protein expression; and by genetic code expansion to add new amino acids to the amino acid alphabet. This review aims to discuss progress in the field of introducing fluorinated handles into biomolecules for NMR studies by post-translational bioconjugation or 'fluorine-tagging'. We will discuss the range of chemical tagging 'warheads' that have been used, explore the applications of fluorine tags, discuss ways to enhance reporter sensitivity and how the signal to noise ratios can be boosted. Finally, we consider some key challenges of the field and offer some ideas for future directions.

Tuning the Reactivity of a Substrate for SNAP-Tag Expands Its Application for Recognition-Driven DNA-Protein Conjugation.

Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs.

A Chemogenetic Approach for the Optical Monitoring of Voltage in Neurons.

Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for the interrogation of the cellular and molecular logic of the nervous system. Herein, a semisynthetic tethered voltage indicator (STeVI1) based upon nile red is described that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and sub-threshold activity in neurons.

Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries.

BACKGROUND: Recombinant peptide chips could constitute a versatile complementation to state-of-the-art in situ (chemical on-chip) synthesis, particle-based printing, or pre-manufactured peptide spotting. Bottlenecks still impeding a routine implementation - from restricted peptide lengths, low diversity and low array densities to high costs - could so be overcome. METHODS: To assess overall performance, we assembled recombinant chips composed of 38,400 individual peptide spots on the area of a standard 96-well microtiter plate from comprehensive, highly diverse (>107 single clones) short random peptide libraries. RESULTS: Screening of altogether 476,160 clones against Streptavidin uncovered 2 discrete new binders: a characteristic HPQ-motif containing VSHPQAPF and a cyclic CSGSYGSC peptide. Interactions were technically confirmed by fluorescence polarization as well as biolayer-interferometry, and their potential suitability as novel detection tags evaluated by detection of a peptide-fused exemplary test protein. CONCLUSION: From our data we conclude that the presented technical pipeline can reliably identify novel hits, useful as first-generation binders or templates for subsequent ligand design plus engineering.

Single Molecule Approaches in RNA-Protein Interactions.

RNA-protein interactions govern every aspect of RNA metabolism, and aberrant RNA-binding proteins are the cause of hundreds of genetic diseases. Quantitative measurements of these interactions are necessary in order to understand mechanisms leading to diseases and to develop efficient therapies. Existing methods of RNA-protein interactome capture can afford a comprehensive snapshot of RNA-protein interaction networks but lack the ability to characterize the dynamics of these interactions. As all ensemble methods, their resolution is also limited by statistical averaging. Here we discuss recent advances in single molecule techniques that have the potential to tackle these challenges. We also provide a thorough overview of single molecule colocalization microscopy and the essential protein and RNA tagging and detection techniques.

Recombinant Intrinsically Disordered Proteins for NMR: Tips and Tricks.

The growing recognition of the several roles that intrinsically disordered proteins play in biology places an increasing importance on protein sample availability to allow the characterization of their structural and dynamic properties. The sample preparation is therefore the limiting step to allow any biophysical method being able to characterize the properties of an intrinsically disordered protein and to clarify the links between these properties and the associated biological functions. An increasing array of tools has been recruited to help prepare and characterize the structural and dynamic properties of disordered proteins. This chapter describes their sample preparation, covering the most common drawbacks/barriers usually found working in the laboratory bench. We want this chapter to be the bedside book of any scientist interested in preparing intrinsically disordered protein samples for further biophysical analysis.

Fluorogenic polymethine dyes by intramolecular cyclization.

Fluorescence imaging plays a pivotal role in the study of biological processes, and cell-permeable fluorogenic dyes are crucial to visualize intracellular structures with high specificity. Polymethine dyes are vitally important fluorophores in single-molecule localization microscopy and in vivo imaging, but their use in live cells has been limited by high background fluorescence and low membrane permeability. In this review, we summarize recent advances in the development of fluorogenic polymethine dyes via intramolecular cyclization. Finally, we offer an outlook on the prospects of fluorogenic polymethine dyes for bioimaging.

Tag-mediated single-step purification and immobilization of recombinant proteins toward protein-engineered advanced materials.

Background: The potential applications of protein-engineered functional materials are so wide and exciting that the interest in these eco-friendly advanced materials will further expand in the future. Tag-mediated protein purification/immobilization technologies have emerged as green and cost-effective approaches for the fabrication of such materials. Strategies that combine the purification and immobilization of recombinant proteins/peptides onto/into natural, synthetic or hybrid materials in a single-step are arising and attracting increasing interest. Aim of Review: This review highlights the most significant advances of the last 5 years within the scope of tag-mediated protein purification/immobilization and elucidates their contributions for the development of efficient single-step purification and immobilization strategies. Recent progresses in the field of protein-engineered materials created using innovative protein-tag combinations and future opportunities created by these new technologies are also summarized and identified herein. Key Scientific Concepts of Review: Protein purification/immobilization tags present a remarkable ability to establish specific non-covalent/covalent interactions between solid materials and biological elements, which prompted the creation of tailor-made and advanced functional materials, and of next-generation hybrid materials. Affinity tags can bind to a wide range of materials (of synthetic, natural or hybrid nature), being most suitable for protein purification. Covalently binding tags are most suitable for long-term protein immobilization, but can only bind naturally to protein-based materials. Hybrid affinity-covalently binding tags have allowed efficient one-step purification and immobilization of proteins onto different materials, as well as the development of innovative protein-engineered materials. Self-aggregating tags have been particularly useful in combination with other tags for generating protein-engineered materials with self-assembling, flexible and/or responsive properties. While these tags have been mainly explored for independent protein purification, immobilization or functionalization purposes, efficient strategies that combine tag-mediated purification and immobilization/functionalization in a single-step will be essential to guarantee the sustainable manufacturing of advanced protein-engineered materials.

Plant Gene Modification by BAC Recombineering.

Recombineering approaches exploiting the bacteriophage λ Red recombination functions are widely used for versatile modification of eukaryotic genes carried by bacterial artificial chromosomes (BACs) in E. coli. Whereas BAC transformation provides a simple means for integration of modified genes into the genomes of animal cells to generate knock-in and knockout lines, successful application of this strategy is hampered by low frequency of homologous recombination in higher plants. However, plant cells can be transformed at a high frequency using the transferred DNA (T-DNA) of Agrobacterium, which is stably and randomly integrated into the plant genome. The function of plant genes that are modified by recombineering and transferred by Agrobacterium T-DNA vectors into plant cells can thus be suitably studied using genetic complementation of knockout mutations induced by either T-DNA insertions or genome editing with T-DNA-based Crisp/Cas9 constructs. Here we describe two recombineering protocols for modification and transfer of plant genes from BACs into Agrobacterium T-DNA plant transformation vectors. The first protocol uses a conditional suicide ccdB gene cassette to assist the genetic complementation assays by generation of point mutations, deletions, and insertions at any gene position. The second "turbo"-recombineering protocol exploits various I-SceI insertion cassettes for fusing of fluorescent protein tags to the plant gene products to facilitate the characterization of their in vivo interacting partners by affinity purification, mass spectrometry, and cellular localization studies.

CRISPR/Cas9-Based Split Fluorescent Protein Tagging.

Genetically encoded fluorescent tags such as green fluorescent protein fused to protein have revolutionized cell biology as they permit high-resolution protein imaging in live systems. Split fluorescent proteins, with a small fragment of 16 amino acids, can be inserted in the coding sequence to label proteins. We demonstrate successful integration of two bright and fast maturing split fluorescent proteins, mNeon green and sfCherry2, in zebrafish, and show that they are suitable for live imaging, including time-lapse series, and that they have a high signal-to-noise ratio. Furthermore, we show that CRISPR/Cas9 can be used to generate fluorescently tagged proteins in vivo.

Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub.

Functional tags are ubiquitous in cell biology, and for studies of one chromosomal locus, the centromere, tags have been remarkably useful. The centromere directs chromosome inheritance at cell division. The location of the centromere is defined by a histone H3 variant, CENP-A. The regulation of the chromatin assembly pathway essential for centromere inheritance and function includes posttranslational modification (PTM) of key components, including CENP-A itself. Others have recently called into question the use of functional tags, with the claim that at least two widely used tags obscured the essentiality of one particular PTM, CENP-AK124 ubiquitination (ub). Here, we employ three independent gene replacement strategies that eliminate large, lysine-containing tags to interrogate these claims. Using these approaches, we find no evidence to support an essential function of CENP-AK124ub. Our general methodology will be useful to validate discoveries permitted by powerful functional tagging schemes at the centromere and other cellular locations.

C-Terminal HA Tags Compromise Function and Exacerbate Phenotypes of Saccharomyces cerevisiae Bloom's Helicase Homolog Sgs1 SUMOylation-Associated Mutants.

The Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which SGS1 was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA). They identified SGS1 mutants that affect its SUMOylation, which we will refer to as SGS1 SUMO-site mutants. In previous work, these mutants showed phenotypes consistent with substantial loss of Sgs1-Top3-Rmi1 function during the mitotic cell cycle. We find that the reported phenotypes are largely due to the presence of the HA epitope tags. Untagged SGS1 SUMO-site mutants show either wild-type or weak hypomorphic phenotypes, depending on the assay. These phenotypes are exacerbated by both 6HA and 3HA epitope tags in two different S. cerevisiae strain backgrounds. Importantly, a C-terminal 6HA tag confers strong hypomorphic or null phenotypes on an otherwise wild-type Sgs1 protein. Taken together, these results suggest that the HA epitope tags used in previous studies seriously compromise Sgs1 function. Furthermore, they raise the possibilities either that sufficient SUMOylation of the Sgs1-Top3-Rmi1 complex might still occur in the SUMO-site mutants isolated, or that Smc5-Smc6-Mms21-mediated SUMOylation plays a minor role in the regulation of Sgs1-Top3-Rmi1 during recombination.

tagPAINT: covalent labelling of genetically encoded protein tags for DNA-PAINT imaging.

Recently, DNA-PAINT single-molecule localization microscopy (SMLM) has shown great promise for quantitative imaging; however, labelling strategies thus far have relied on multivalent and affinity-based approaches. Here, the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA-docking strands and application of SMLM via DNA-PAINT is demonstrated. tagPAINT is then used for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

Pick a Tag and Explore the Functions of Your Pet Protein.

Protein tags have been essential for advancing our knowledge of the function of proteins, their localization, and the mapping of their interaction partners. Expressing epitope-tagged proteins has become a standard practice in every life science laboratory and, thus, continues to enable new studies. In recent years, several new tagging moieties have entered the limelight, many of them bringing new functionalities, such as targeted protein degradation, accurate quantification, and proximity labeling. Other novel tags aim at tackling research questions in challenging niches. In this review, we elaborate on recently introduced tags and the opportunities they provide for future research endeavors. In addition, we highlight how the genome-engineering revolution may boost the field of protein tags.

Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation.

4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.

Elucidating the role of an immunomodulatory protein in cancer: From protein expression to functional characterization.

Several fundamental discoveries made over the last two decades, in the field of cancer biology, have increased our understanding of the complex tumor micro- and macroenvironments. This has shifted the current empirical cancer therapies to more rationalized treatments targeting immunomodulatory proteins. From the point of identification, a protein target undergoes several interrogations, which are necessary to truly define its druggability. Here, we outline some basic steps that can be followed for in vitro characterization of a potential immunomodulatory protein target. We describe procedures for recombinant protein expression and purification including key annotations on protein cloning, expression systems, purification strategies and protein characterization using structural and biochemical approaches. For functional characterization, we provide detailed protocols for using flow-cytometric techniques in cell lines or primary cells to study protein expression profiles, proliferation, apoptosis and cell-cycle changes. This multilevel approach can provide valuable, in-depth understanding of any protein target with potential immunomodulatory effects.

Analysis of the Localization of MEN Components by Live Cell Imaging Microscopy.

Mitotic exit is determined by multiple spatial and temporal cues from the spindle poles and the two compartments in a dividing yeast cell-the mother and the bud. These signals are ultimately integrated by the activation of the mitotic exit network (MEN) to promote persistent release of Cdc14 from the nucleolus. Live imaging analysis using fluorescent protein tags is invaluable to dissect this critical decision-making trigger. Here, we present protocols for routine yeast live cell microscopy applicable to this problem.

Purification of Proteins Fused to Maltose-Binding Protein.

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

Protein Affinity Purification using Intein/Chitin Binding Protein Tags.

Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protein of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.