Search

Site-specific incorporation of artificial functionalities into protein targets is an important tool in both basic and applied research and can be a major challenge to protein chemists. Chemical labeling methods often targeting multiple positions within a protein and therefore suffer from lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this contribution we describe the application of the highly specific trypsin variant named trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Both reaction regimes proceed under mild and bioorthogonal conditions in a short period of time which result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the construction of peptide or acyl donor ester probes used as substrates for the biocatalyst. In addition, we provide instructions how to perform the ultimate bioconjugation reactions and finally render assistance for the qualitative and quantitative analysis of the reaction course and outcome.

The advent of new DNA sequencing technologies leads to a dramatic increase in the number of available genome sequences and therefore of target genes with potential for functional analysis. The insertion of these sequences into proper expression vectors requires a simple an efficient cloning method. In addition, when expressing a target protein, quite often it is necessary to evaluate different DNA constructs to achieve a soluble and homogeneous expression of the target with satisfactory yields. The development of new molecular methods made possible the cloning of a huge number of DNA sequences in a high-throughput manner, necessary for meeting the increasing demands for soluble protein expression and characterization. In this chapter several molecular methods suitable for high-throughput cloning are reviewed.

Cystatin C (CST3) is expressed ubiquitously and implicated in several neurological diseases. It can be posttranscriptionally modified. CST3 is usually quantified in a biological sample using antibody-based methods. Posttranscriptional modification can hamper antibody-based detection systems by altering antibody-binding epitope(s). To circumvent this problem, enzymatic digestion and liquid chromatography tandem mass spectrometry (LC-MS/MS) technique can be employed to identify and measure peptides of a target protein in a complex biological mixture. This chapter describes an LC-MS/MS-based method for accurate measurement of CST3 in cerebrospinal fluid (CSF). Here, CSF was directly subjected to trypsin digestion and digested peptides were extracted using a solid-phase extraction column. Extracted peptide samples were directly used for LC-MS/MS-based identification and quantification of CST3 peptides. Comparing the concentration in a set of samples measured by LC-MS/MS with that of immunoassay shows that it was significantly higher when measured by LC-MS/MS method, suggesting it a better quantification method. This approach is particularly well suited when posttranscriptional modification of CST3 is suspected and sample volume of CSF is small.

Site-specific incorporation of nonproteinogenic functionalities into protein targets is an important tool in both basic and applied research and represents a major challenge to protein chemists. Chemical labeling methods often target multiple positions within a protein and therefore suffer from a lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this chapter we describe the application of the highly specific trypsin variant trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Reactions rapidly proceed under mild conditions and result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the synthesis of peptide (ester) substrates. In addition, we provide instructions for the bioconjugation reactions and for the qualitative and quantitative analysis of reaction progress and efficiency.

Flow-Induced Dispersion Analysis (FIDA) enables characterization and quantification of proteins under native conditions. FIDA is based on measuring the change in size of a ligand as it selectively interacts with the target protein. The unbound ligand has a relatively small apparent hydrodynamic radius (size), which increase in the presence of the analyte due to binding to the analyte. The Kd of the interaction may be obtained in a titration experiment and the measurement of the apparent ligand size in an unknown sample forms the basis for determining the analyte concentration. The apparent molecular size is measured by Taylor dispersion analysis (TDA) in fused silica capillary capillaries. FIDA is a "ligand-binding" assay and has therefore certain features in common with Enzyme-Linked Immunosorbent Assay (ELISA), Surface Plasmon Resonance (SPR), and Biolayer Interferometry (BLI) based techniques. However, FIDA probes a single in-solution binding event and thus makes assay development straightforward, and the absolute size measurement enables built-in assay quality control. Further, as FIDA does not involve surface chemistries, complications related to nonspecific adsorption of analyte and assay components are minimized enabling direct measurement in, e.g., plasma and serum.

Pichia pastoris is a common host organism for the production of recombinant proteins. While unglycosylated recombinant proteins derived from this yeast can be purified efficiently by only a few conventional chromatography steps, the purification of glycosylated recombinant proteins is a very challenging process due to the intrinsic feature of the yeast of hypermannosylation. The resulting vast glycosylation pattern on the recombinant target protein masks its physicochemical properties hampering a conventional downstream process. Here, we describe a fast and efficient two-step chromatography strategy, where both steps are operated in flow-through mode, to purify recombinant glycoproteins from P. pastoris culture supernatants.

Protein bioconjugation benefits from precise regional and temporal control. One notable way of achieving this control is through the enzymatic attachment of bioorthogonal reactive handles to peptide recognition sequences that are genetically fused to target proteins of interest. The lipoic acid ligase variant, LplAW37V, functionalizes proteins by covalently attaching an azide-bearing lipoic acid derivative to a 13-amino acid recognition sequence known as the lipoic acid ligase acceptor peptide (LAP). Once attached, the azide group can be modified with diverse chemical entities through azide-alkyne click chemistry, enabling conjugation of chemical probes such as fluorophores and facilitating polymer attachment, glycosylation, and protein immobilization in addition to many other possible chemical modifications. The versatility of the attached azide group is complemented by the modular nature of the LAP sequence, which can be introduced within a protein at internal and/or terminal sites as well as at multiple sites simultaneously. In this chapter we describe the in vitro LplAW37V-mediated ligation of 10-azidodecanoic acid to a LAP-containing target protein (i.e., green fluorescent protein (GFP)) and the characterization of the ligation reaction products. Additionally, methods for the modification and immobilization of azide-functionalized LAP-GFP are discussed.

Immunohistochemistry (IHC) is a commonly used technique for protein detection in tissue sections. The method requires high-affinity antibodies that are specific for the target proteins of interest. More advanced IHC techniques have been developed to meet the need for simultaneous detection of more than one target protein in the same tissue section. This chapter provides general guidelines for double IHC staining of formalin-fixed, paraffin-embedded tissue sections. Chromogenic substrates are chosen based on their excellent contrast and compatibility with the subsequent digital image analysis.

Affinity agents coupled to a functional moiety play an ever-increasing role in modern medicine, ranging from radiolabeled selective binders in diagnosis to antibody-drug conjugates in targeted therapies. In biomedical research, protein coupling to fluorophores, surfaces and nanoparticles has become an integral part of many procedures. In addition to antibodies, small scaffold proteins with similar target binding properties are being widely explored as alternative targeting moieties. To label these binders of interest with different functional moieties, conventional chemical coupling methods can be employed, but often result in heterogeneously modified protein products. In contrast, enzymatic labeling methods are highly site-specific and efficient. Protein farnesyltransferase (PFTase) catalyzes the transfer of an isoprenoid moiety from farnesyl diphosphate (FPP) to a cysteine residue in a C-terminal CaaX motif at the C-terminus of a protein substrate. The addition of only four amino acid residues minimizes the influence on the native protein structure. In addition, a variety of isoprenoid analogs containing different bioorthogonal functional groups, including azides, alkynes, and aldehydes, have been developed to enable conjugation to various cargos after being incorporated onto the target protein by PFTase. In this protocol, we present a detailed procedure for labeling Designed Ankyrin Repeat Proteins (DARPins) engineered with a C-terminal CVIA sequence using an azide-containing FPP analog by yeast PFTase (yPFTase). In addition, procedures to subsequently conjugate the labeled DARPins to a TAMRA fluorophore using strained-promoted alkyne-azide cycloaddition (SPAAC) reactions as well as the sample preparation to evaluate the target binding ability of the conjugates by flow cytometry are described.

This chapter will focus on today's in silico direct and indirect approaches to assess therapeutic target druggability. The direct approach tries to infer from the 3D structure the capacity of the target protein to bind small molecule in order to modulate its biological function. Algorithms to recognize and characterize the quality of the ligand interaction sites whether within buried protein cavities or within large protein-protein interface will be reviewed in the first part of the paper. In the case a ligand-binding site is already identified, indirect aspects of target druggability can be assessed. These indirect approaches focus first on target promiscuity and the potential difficulties in developing specific drugs. It is based on large-scale comparison of protein-binding sites. The second aspect concerns the capacity of the target to induce resistant pathway once it is inhibited or activated by a drug. The emergence of drug-resistant pathways can be assessed through systemic analysis of biological networks implementing metabolism and/or cell regulation signaling.

Posttranslational modifications (PTMs) happen after or during protein translation. Small Ubiquitin-like Modifier (SUMO) proteins are covalently attached to certain lysine residues of the target proteins to modify their activity, stability, or localization. This process is called SUMOylation, which is a reversible PTM: SUMO protease enzymes can cleave SUMOs off the target protein backbone. Although many ubiquitinated proteins are targeted for degradation, SUMOylation does not necessary lead to the degradation of the modified protein but lead to the regulation of various physiological responses. SUMOylation of the examined protein cannot simply be monitored by immunoblotting techniques performed on total protein extracts, due to the SUMO-specific signals derived from other modified molecules. Furthermore, the fact that only a limited fraction of the target protein pool is SUMOylated makes the detection of SUMOylated proteins challenging. This protocol shows how SUMOylated phytochrome B (phyB) molecules can be detected using homologous and heterologous experimental systems in planta.

After injecting their genome into the bacterial host cell, bacteriophages need to convert the host metabolism toward efficient phage production. For this, specific proteins have evolved which interact with key host proteins to inhibit, activate or redirect the function of these proteins. Since 70% of the currently annotated phage genes are hypothetical proteins of unknown function, the identification and characterization of these phage proteins involved in host-phage protein-protein interactions remains challenging. Here, we describe a method to identify phage proteins involved in host-phage protein-protein interactions using a combination of affinity purifications and mass spectrometry analyses. A bacterial strain is engineered in which a bacterial target protein is fused to a Strep-tag® II at the C-terminal end. This strain is infected with a specific bacteriophage, followed by an affinity purification of the tagged protein which allows the copurification of all bacterial and phage specific interacting proteins. After SDS-PAGE analysis and an in-gel trypsin digestion, the purified interacting proteins are identified by mass spectrometry analysis. The identification of phage proteins involved in interactions provides first hints toward the elucidation of the biological function of these proteins.

Label-free porous silicon (PSi)-based biosensor enables the detection and identification of the protein in transgenic cotton. Changes in optical response signal in the presence of the target protein can be detected by Fourier transform infrared (FTIR) spectromicroscopy when binding of the target protein with its antibody is selectively captured on the PSi biosensor. Here we describe the transgenic protein (antifreeze protein, AFP), and compare with non-transgenic plants. Significant red shifts are observed for transgenic antifreeze protein cotton lines.

Many small molecule compounds with anticancer activities are discovered through phenotype-based screens. However, discovering the targets of these small molecules has been challenging. The gold standard for target identification requires the discovery of mutations in the target protein that block the effects of small molecules in vitro as well as in vivo. Here we describe the procedures for isolating drug resistant clones using the colorectal cancer cell line HCT-116 followed by whole-exome sequencing to identify recurrent mutations associated with compound resistance. Together with downstream in vitro and in vivo validation experiments, this strategy enables rapid target discovery for cytotoxic compounds.

Biological activities are mainly executed by proteins and in most of the occasions these activities are accomplished by protein complexes or through protein-protein interactions (PPI). So it is critical to reveal how the protein complexes are organized and demonstrate the PPIs involved in the biological processes. In addition to the traditional biochemical approaches, proximity-dependent labeling (PDL) has recently been proposed to identify the interacting partners of a given protein. PDL requires the fusion expression of the target protein with an enzyme which catalyzes the attachment of a reactive molecule to the interacting partners in a distance-dependent manner. Further analysis of all the proteins that are modified by the reactive molecule discloses the identity of these proteins which are presumed to be interacting partners of the target protein. BioID is one of those representative PDL methods with the most widely applications. The enzyme used in BioID is a biotin ligase BirA which catalyzes the biotinylation of target protein with the presence of biotin. Through streptavidin-mediated pull-down and mass spectrometry analysis, the interacting protein candidates of a given protein can be obtained.

About 20% of the cancer incidences worldwide have been estimated to be associated with infections. However, the molecular mechanisms of exactly how they contribute to host tumorigenesis are still unknown. To evade host defense, pathogens hijack host proteins at different levels: sequence, structure, motif, and binding surface, i.e., interface. Interface similarity allows pathogen proteins to compete with host counterparts to bind to a target protein, rewire physiological signaling, and result in persistent infections, as well as cancer. Identification of host-pathogen interactions (HPIs)-along with their structural details at atomic resolution-may provide mechanistic insight into pathogen-driven cancers and innovate therapeutic intervention. HPI data including structural details is scarce and large-scale experimental detection is challenging. Therefore, there is an urgent and mounting need for efficient and robust computational approaches to predict HPIs and their complex (bound) structures. In this chapter, we review the first and currently only interface-based computational approach to identify novel HPIs. The concept of interface mimicry promises to identify more HPIs than complete sequence or structural similarity. We illustrate this concept with a case study on Kaposi's sarcoma herpesvirus (KSHV) to elucidate how it subverts host immunity and helps contribute to malignant transformation of the host cells.

With 49 members identified thus far, the superfamily of nuclear receptors offers a large number of targets to be pharmacologically exploited. Some nuclear receptors already look back to a successful history as drug targets, while others still lack any identified ligand. The development of small molecules targeting nuclear receptor is a challenging task and has to consider not only high affinity binding but also aspects as the nuclear localization of the target protein or transactivation efficacy. In this chapter, we summarize characteristics of nuclear receptors as target family, strategies of hit and lead identification, and the variety of methods for in vitro characterization of nuclear receptor modulators. A detailed method chapter describes an example optimization of a nuclear receptor modulator as well as hybrid reporter gene assays as a very flexible method of choice for in vitro characterization. Thereby, the chapter provides an introduction to nuclear receptor ligand development.

This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA. A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identified which corresponded to a 20 mer sequence of the VapA protein.

Chromatin immunoprecipitation (ChIP), originally developed by John T. Lis and David Gilmour in 1984, has been useful to detect DNA sequences where protein(s) of interest bind. ChIP is comprised of several steps: (1) cross-linking of proteins to target DNA sequences, (2) breaking genomic DNA into 300-1000 bp pieces by sonication or nuclease digestion, (3) immunoprecipitation of protein bound to target DNA with an antibody, (4) reverse cross-linking between target DNA and the bound protein to liberate the DNA fragments, and (5) amplification of target DNA fragment by PCR. Since then, the technology has evolved significantly to allow not only amplifying target sequences by PCR, but also sequencing all DNA fragment bound to a target protein, using a variant of the approach called the ChIP-seq technique (1). Another variation, the ChIP-on-ChIP, allows the detection of protein complexes bound to specific DNA sequences (2).

Immunoprecipitation is one of the most important and widely used techniques for the detection and purification of a protein of interest. Thanks to highly specific interaction between antigen and antibody, a target protein is purified and concentrated effectively. To obtain reasonable amounts of a target protein, it is crucially important to prepare total cell lysates in which the target protein is present in a soluble form. Here, we describe methods to prepare total cell lysates of fission yeast, which are then used directly for immunoprecipitation. We also describe some tips to select reagents for preparing buffers having a substantial impact on protein solubility, because there is essentially no reagent that can accommodate the full range of proteins having different characteristics.

Super-resolution microscopy allows imaging of cellular structures at nanometer resolution. This comes with a demand for small labels which can be attached directly to the structures of interest. In the context of protein labeling, one way to achieve this is by using genetic code expansion (GCE) and click chemistry. With GCE, small labeling handles in the form of noncanonical amino acids (ncAAs) are site-specifically introduced into a target protein. In a subsequent step, these amino acids can be directly labeled with small organic dyes by click chemistry reactions. Click chemistry labeling can also be combined with other methods, such as DNA-PAINT in which a "clickable" oligonucleotide is first attached to the ncAA-bearing target protein and then labeled with complementary fluorescent oligonucleotides. This protocol will cover both aspects: I describe (1) how to encode ncAAs and perform intracellular click chemistry-based labeling with an improved GCE system for eukaryotic cells and (2) how to combine click chemistry-based labeling with DNA-PAINT super-resolution imaging. As an example, I show click-PAINT imaging of vimentin and low-abundance nuclear protein, nucleoporin 153.

Rational drug design is essential for new drugs to emerge, especially when the structure of a target protein or catalytic enzyme is known experimentally. To that purpose, high-throughput virtual ligand screening campaigns aim at discovering computationally new binding molecules or fragments to inhibit a particular protein interaction or biological activity. The virtual ligand screening process often relies on docking methods which allow predicting the binding of a molecule into a biological target structure with a correct conformation and the best possible affinity. The docking method itself is not sufficient as it suffers from several and crucial limitations (lack of protein flexibility information, no solvation effects, poor scoring functions, and unreliable molecular affinity estimation).At the interface of computer techniques and drug discovery, molecular dynamics (MD) allows introducing protein flexibility before or after a docking protocol, refining the structure of protein-drug complexes in the presence of water, ions and even in membrane-like environments, and ranking complexes with more accurate binding energy calculations. In this chapter we describe the up-to-date MD protocols that are mandatory supporting tools in the virtual ligand screening (VS) process. Using docking in combination with MD is one of the best computer-aided drug design protocols nowadays. It has proved its efficiency through many examples, described below.

The unique property of specific high affinity binding to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence, knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe an updated protocol for high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric analysis. Epitopes are determined by DNA sequencing of the sorted antibody-binding cells followed by sequence alignment back to the antigen sequence. The method described here has been useful for the mapping of both monoclonal and polyclonal antibodies with varying sizes of epitopes.

Virtual screening is a computational technique for predicting a potent binding compound for a receptor protein from a ligand library. It has been a widely used in the drug discovery field to reduce the efforts of medicinal chemists to find hit compounds by experiments.Here, we introduce our novel structure-based virtual screening program, PL-PatchSurfer, which uses molecular surface representation with the three-dimensional Zernike descriptors, which is an effective mathematical representation for identifying physicochemical complementarities between local surfaces of a target protein and a ligand. The advantage of the surface-patch description is its tolerance on a receptor and compound structure variation. PL-PatchSurfer2 achieves higher accuracy on apo form and computationally modeled receptor structures than conventional structure-based virtual screening programs. Thus, PL-PatchSurfer2 opens up an opportunity for targets that do not have their crystal structures. The program is provided as a stand-alone program at http://kiharalab.org/plps2 . We also provide files for two ligand libraries, ChEMBL and ZINC Drug-like.

Knowledge of the exquisite-binding surface of an antibody on its target protein is of great value, in particular for therapeutic antibodies for understanding method of action and for stratification of patients carrying the necessary epitope for desired drug efficacy, but also for capture assays under native conditions. Several epitope mapping methodologies have been described for this purpose, with the laborious X-ray crystallography method being the ideal method for mapping of discontinuous epitopes in antibody-antigen crystal complexes and high-throughput peptide-based methods for mapping of linear epitopes. We here report on the usage of a bacterial surface display-based method for mapping of structural epitopes by display of folded domains on the surface of Gram positive bacteria, followed by domain-targeted mutagenesis and library analysis for the identification of key-residues by flow sorting and sequencing. Identified clones with reduced affinity are validated by single clone FACS and subsequent full-length expression in mammalian cells for validation.

Single-domain antibody fragments, also called nanobodies (Nbs), are increasingly being used as targeting molecular tools for imaging and/or targeted radionuclide therapy. To translate these tools to the clinic, it is preferred to obtain a homogeneous, well-defined, and well-characterized product. It has been shown that Sortase A, a transpeptidase found in Staphylococcus aureus, catalyzes the site-specific conjugation between a recognition oligopeptide (LPXTG, known as sortag) and an oligoglycine functionalized probe. This versatile technique manages to couple various molecular reagents, such as biotin, fluorophores, bifunctional chelators, etc., to the target protein containing the sortag. This chapter focuses on the site-specific coupling of a bifunctional chelator (e.g., CHX-A"-DTPA) to a Nb equipped with a C-terminal sortag. The chelator conjugated to the Nb can be radiolabeled with 111In or 177Lu for SPECT imaging or targeted radionuclide therapy, respectively.

Sequence-specific nucleases (SSNs) are nowadays fundamental tools to generate mutants that impaired in genes of interest. The bioactive molecules screened in the chemical genomics studies affect specific physiological process by disrupting the function of its target protein(s). Mutation analysis of the gene(s) of target protein(s) of the screened chemical is necessary to resolve how the chemical works in plants. Clustered regularly interspersed short palindromic repeats (CRISPR) from Prevotella and Francisella 1 (Cpf1) are newly characterized RNA-directed endonuclease. Several papers have shown clearly that Cpf1 could be a versatile SSN in plant genome engineering. Cfp1 from Francisella novicida (FnCpf1) recognizes TTN as its protospacer adjacent motif (PAM). FnCpf1 utilizes a shorter PAM compared to other known Cpf1s such as AsCpf1 or LbCpf1, which use TTTN as PAM. Since PAM length can be a limiting factor in target selection, this feature of FnCpf1 is practical for targeted mutagenesis experiments. The application of FnCpf1-mediated targeted mutagenesis to the chemical genomics could accelerate to figure out the mechanism of action of screened chemicals. Here, we describe procedures for targeted mutagenesis in rice and tobacco using FnCpf1.

Ion channels represent nearly a quarter of all targets that currently available medications modulate, and their dysfunction underlies increasing number of human diseases. Functional analysis of ion channels have traditionally been a bottleneck in large-scale analyses. Recent technological breakthroughs in automated planar electrophysiology have democratized the technique to enable high-throughput patch clamping at scale. In this chapter, we describe the methodology to perform a phenotypic screen on voltage-gated calcium channels across many different genetic coding variations and against small-molecule modulators. We first describe the procedures to establish inducible heterologous ion channel expression in HEK293 cells, where each cell incorporates one copy of a target protein cDNA-a step that is critical for producing stable and consistent expression of ion channels. We then describe the experimental and analytical methods for analyzing the function of ion channels using high-throughput planar electrophysiology.

The synthetic photoaffinity probe designed to mimic bioactive molecules is one of the powerful tools for the identification of the target protein in living organisms. However, nonspecific interaction between the probe and nontargets would cause a misleading result in many cases of the photoaffinity labeling. In this chapter, we describe an enantiodifferential approach as a reliable method for the detection of the specific target protein of the bioactive natural product, jasmonate glucoside, a chemical factor that controls the nyctinastic leaf movement of the leguminous plants.

Western blotting is a common approach to detect the presence of a target protein in biological samples or proteins mixture using specific antibodies. This method is also useful to study regulation of virulence determinants by analyzing changes in protein expression between different genetic backgrounds or under varying environmental conditions. To avoid the need to raise specific antibodies for each studied protein, commercial antibody against commonly used peptidic epitopes can be utilized if the right target tagged version is constructed. Here we describe a C-terminal fusion between a protein of interest and the two hemagglutinin A (2HA) tag. The tagged protein is cloned into a low-copy number vector and expressed under its native promoter in Salmonella enterica. Then, the expression of the tagged protein can be analyzed by Western blotting and commercially available anti-2HA antibodies.

The PARP family of ADP-ribosyl transferases contains 17 members in human cells, most of which catalyze the transfer of the ADP-ribose moiety of NAD(+) onto their target proteins. This post-translational modification plays important roles in cellular signaling, especially during cellular stresses, such as heat shock, inflammation, unfolded protein responses, and DNA damage. Knowing the specific proteins that are substrates for individual PARPs, as well as the specific amino acid residues in a give target protein that are ADP-ribosylated, is a key step in understanding the biology of individual PARPs. Recently, we developed a robust NAD(+) analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition (‘click chemistry’) reactions. When coupled with proteomics and mass spectrometry, the analog-sensitive PARP approach can be used to identify the specific amino acids that are ADP-ribosylated by individual PARP proteins. In this chapter, we describe the key facets of the experimental design and application of the analog-sensitive PARP methodology to identify site-specific modification of PARP target proteins.

Giardia lamblia, a major parasite, is an emerging model organism due to its compact genomic arrangement and composition. The most popular reverse genetic technique, RNAi, is ineffective in Giardia. In contrast, protein depletion by translation blocking morpholinos is suitable for most gene targets and provides up to 80% depletion of the target protein. The method is fast, reliable, and specific. After antisense morpholino oligomer delivery into Giardia trophozoites by electroporation, the cells can be used for many subsequent analyses 8-48 h after treatment. In this chapter, suitable gene tags, plasmids, and techniques necessary for proper morpholino targeting are described.

We introduce a web server called GalaxyPepDock that predicts protein-peptide interactions based on templates. With the continuously increasing size of the protein structure database, the probability of finding related proteins for templates is increasing. GalaxyPepDock takes a protein structure and a peptide sequence as input and returns protein-peptide complex structures as output. Templates for protein-peptide complex structures are selected from the structure database considering similarity to the target protein structure and to putative protein-peptide interactions as estimated by protein structure alignment and peptide sequence alignment. Complex structures are then built from the template structures by template-based modeling. By further structure refinement that performs energy-based optimization, structural aspects that are missing in the template structures or that are not compatible with the given protein and peptide are refined. During the refinement, flexibilities of both protein and peptide induced by binding are considered. The atomistic protein-peptide interactions predicted by GalaxyPepDock can offer important clues for designing new peptides with desired binding properties.

Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.

Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD+) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.

Molecular and chemical chaperones /foldases can strongly contribute to improve the amounts and the structural quality of recombinant proteins. Several methodologies have been proposed to optimize their beneficial effects. This chapter presents a condensed summary of the biotechnological opportunities offered by this approach followed by a protocol describing the method we use for expressing disulfide bond-dependent recombinant antibodies in the cytoplasm of bacteria engineered to overexpress sulfhydryl oxidase and DsbC isomerase. The system is based on the possibility to trigger the foldase expression independently and before the induction of the target protein. As a consequence, the recombinant antibody synthesis starts only after enough foldases have accumulated to promote correct folding of the antibody.

Recent advancements in mass spectrometry (MS) and data analysis software have enabled new strategies for biological discovery using proteomics. Proteomics has evolved from routine discovery and identification of proteins to integrated multi-omics projects relating specific proteins to their genes and metabolites. Using additional information, such as that contained in biological pathways, has enabled the use of targeted protein quantitation for monitoring fold changes in expression as well as biomarker discovery. Here we discuss a full proteomic workflow from discovery proteomics on a quadrupole Time-of-Flight (Q-TOF) MS to targeted proteomics using a triple quadrupole (QQQ) MS. A discovery proteomics workflow encompassing acquisition of data-dependent proteomics data on a Q-TOF and protein database searching will be described which uses the protein abundances from identified proteins for subsequent statistical analysis and pathway visualization. From the active pathways, a protein target list is created for use in a peptide-based QQQ assay. These peptides are used as surrogates for target protein quantitation. Peptide-based QQQ assays provide sensitivity and selectivity allowing rapid and robust analysis of large batches of samples. These quantitative results are then statistically compared and visualized on the original biological pathways with a more complete coverage of proteins in the studied pathways.

The increasing number of peptide and protein biomarker candidates requires expeditious and reliable quantification strategies. The utilization of liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) for the absolute quantitation of plasma proteins and peptides facilitates the multiplexed verification of tens to hundreds of biomarkers from smallest sample quantities. Targeted proteomics assays derived from bottom-up proteomics principles rely on the identification and analysis of proteotypic peptides formed in an enzymatic digestion of the target protein. This protocol proposes a procedure for the establishment of a targeted absolute quantitation method for middle- to high-abundant plasma proteins waiving depletion or enrichment steps. Essential topics as proteotypic peptide identification and LC-MS/MS method development as well as sample preparation and calibration strategies are described in detail.

Modulation of signal transduction via binding of the secondary messenger molecule cyclic di-GMP to effector proteins is a near universal regulatory schema in bacteria. In particular, direct binding of c-di-GMP to transcriptional regulators has been shown to alter gene expression of a variety of processes. Here, we illustrate a pull-down-based DNA:protein binding reaction to determine the relative importance of c-di-GMP in the binding affinity of a target protein to specific DNA sequences. Specifically, the pull-down-based assay enables DNA binding to be analyzed with differing concentrations of c-di-GMP in the absence/presence of specific and nonspecific competitors.

Recombinant expression of disulfide-reticulated peptides and proteins is often challenging. We describe a method that exploits the periplasmic disulfide-bond forming machinery of Escherichia coli and combines this with a cleavable, solubility-enhancing fusion tag to obtain higher yields of correctly folded target protein than is achievable via cytoplasmic expression. The protocols provided herein cover all aspects of this approach, from vector construction and transformation to purification of the cleaved target protein and subsequent quality control.

Recombinant expression of heterologous proteins in E. coli is well established for a wide range of proteins, although in many cases, purifying soluble and properly folded proteins remains challenging (Sorensen and Mortensen, J Biotechnol 115:113-128, 2005; Correa and Oppezzo, Methods Mol Biol 1258:27-44, 2015). Proteins that contain disulfide bonds (e.g., cytokines, growth factors) are often particularly difficult to purify in soluble form and still need optimizing of protocols in almost every step of the process (Berkmen, Protein Expr Purif 82:240-251, 2012; de Marco, Microb Cell Fact 11:129, 2012). Expression of disulfide bonded proteins in the periplasm of E. coli is one approach that can help to obtain soluble protein with the correct disulfide bridges forming in the periplasm. This offers the appropriate conditions for disulfide formation although periplasmic expression can also result in low expression levels and incorrect folding of the target protein (Schlapschy and Skerra, Methods Mol Biol 705:211-224, 2011). Generation of specific antibodies often requires a specific antigenic sequence of a protein in order to get an efficient immune response and minimize cross-reactivity of antibodies. Larger proteins like GST (Glutathione-S-transferase) or MBP (maltose binding protein) as solubilizing fusion partners are frequently used to keep antigens soluble and immunize animals. This approach has the disadvantage that the immune response against the fusion partner leads to additional antibodies that need to be separated from the antigen-specific antibodies. For both classes of proteins mentioned above, a protocol has been developed and optimized using the human version of small ubiquitin-like modifier 3 (SUMO3) protein and its corresponding protease SenP2. This chapter describes the experimental steps for expression, purification, refolding, and cleavage that are applicable to both disulfide-bonded proteins with a defined structure and random protein fragments for antibody generation or larger peptides with defined sequence that are difficult express on their own.

Molecular replacement (MR), a method for solving the crystallographic phase problem using phases derived from a model of the target structure, has proven extremely valuable, accounting for the vast majority of structures solved by X-ray crystallography. However, when the resolution of data is low, or the starting model is very dissimilar to the target protein, solving structures via molecular replacement may be very challenging. In recent years, protein structure prediction methodology has emerged as a powerful tool in model building and model refinement for difficult molecular replacement problems. This chapter describes some of the tools available in Rosetta for model building and model refinement specifically geared toward difficult molecular replacement cases.

Expression of recombinant proteins in sufficient quantities is essential for protein structure-function studies. The most commonly used method for recombinant protein production is overexpression in E. coli cultures. However, producing high yields of functional proteins in E. coli can be a challenge in conventional shaken cultures. This is often due to nonoptimal growth conditions, which result in low cell yields and insoluble or incorrectly folded target protein. To overcome the shortcomings of shake flask cultivation, we present a cultivation method based on enzymatic glucose delivery. This system mimics the fed-batch principle used in bioreactor cultivations and provides high yields of biomass and recombinant proteins in shaken cultivations.

Conditional modulation of biological processes plays key roles in basic and applied research and in translation. It can be achieved on various levels via a multitude of approaches. One of the directions is manipulating target protein levels and activity by transcriptional, posttranscriptional, translational, and posttranslational control. Because in most of these techniques, the synthesis of the target proteins is adjusted to the needs, they all rely on the specific half-life of the target protein and its turn-over. Therefore, their time-of-action, in direct correlation to the desired reprogramming of molecular phenotypes caused by altering the target levels, is fixed and determined by the naturally inherent properties. We have introduced the low-temperature degron (lt-degron) to various intact multicellular organisms which allows to control target protein levels and therefore function and activity directly on the level of active protein. The lt-degron uses a combination of Ubiquitin-fusion technique linking target protein degradation to the N-end rule pathway of targeted proteolysis coupled with the use of cell- and tissue-specific promoters.

Alternate frame folding (AFF) and protein/fragment exchange (FREX) are related technologies for engineering allosteric conformational changes into proteins that have no pre-existing allosteric properties. One of their chief purposes is to turn an ordinary protein into a biomolecular switch capable of transforming an input event into an optical or functional readout. Here, we present a guide for converting an arbitrary binding protein into a fluorescent biosensor with Förster resonance energy transfer output. Because the AFF and FREX mechanisms are founded on general principles of protein structure and stability rather than a property that is idiosyncratic to the target protein, the basic design steps—choice of permutation/cleavage sites, molecular biology, and construct optimization—remain the same for any target protein. We highlight effective strategies as well as common pitfalls based on our experience with multiple AFF and FREX constructs.