Isothermal titration calorimetry (ITC) is a powerful method for studying protein–DNA interactions in solution. As long as binding is accompanied by an appreciable enthalpy change, ITC studies can yield quantitative information on stoichiometries, binding energetics (affinity, binding enthalpy and entropy) and potential site–site interactions (cooperativity). This can provide a full thermodynamic description of an interacting system which is necessary to understand the stability and specificity of protein–DNA interactions and to correlate the activities or functions of different species. Here we describe procedures to perform and analyze ITC studies using as examples, the E. coli SSB (homotetramer with 4 OB-folds) and D. radiodurans SSB (homodimer with 4 OB-folds). For oligomeric protein systems such as these, we emphasize the need to be aware of the likelihood that solution conditions will influence not only the affinity and enthalpy of binding but also the mode by which the SSB oligomer binds ssDNA.
EcoSSB; DrSSB; ssDNA binding; ITC; SSB-ssDNA thermodynamics
The field of metabolomics has witnessed an exponential growth in the last decade driven by important applications spanning a wide range of areas in the basic and life sciences and beyond. Mass spectrometry in combination with chromatography and nuclear magnetic resonance are the two major analytical avenues for the analysis of metabolic species in complex biological mixtures. Owing to its inherent significantly higher sensitivity and fast data acquisition, MS plays an increasingly dominant role in the metabolomics field. Propelled by the need to develop simple methods to diagnose and manage the numerous and widespread human diseases, mass spectrometry has witnessed tremendous growth with advances in instrumentation, experimental methods, software, and databases. In response, the metabolomics field has moved far beyond qualitative methods and simple pattern recognition approaches to a range of global and targeted quantitative approaches that are now routinely used and provide reliable data, which instill greater confidence in the derived inferences. Powerful isotope labeling and tracing methods have become very popular. The newly emerging ambient ionization techniques such as desorption ionization and rapid evaporative ionization have allowed direct MS analysis in real time, as well as new MS imaging approaches. While the MS-based metabolomics has provided insights into metabolic pathways and fluxes, and metabolite biomarkers associated with numerous diseases, the increasing realization of the extremely high complexity of biological mixtures underscores numerous challenges including unknown metabolite identification, biomarker validation, and interlaboratory reproducibility that need to be dealt with for realization of the full potential of MS-based metabolomics. This chapter provides a glimpse at the current status of the mass spectrometry-based metabolomics field highlighting the opportunities and challenges.
Mass spectrometry; Ionization methods; Quantitative metabolomics; Mass analyzers; Ambient ionization; MS-imaging; Chromatography; Capillary electrophoresis
NADH is an essential redox cofactor in numerous metabolic reactions, and the cytosolic NADH-NAD+ redox state is a key parameter in glycolysis. Conventional NADH measurements rely on chemical determination or autofluorescence imaging, which cannot assess NADH specifically in the cytosol of individual live cells. By combining a bacterial NADH-binding protein and a fluorescent protein variant, we have created a genetically encoded fluorescent biosensor of the cytosolic NADH-NAD+ redox state, named Peredox (Hung et al., Cell Metab 14:545–554, 2011). Here, we elaborate on imaging methods and technical considerations of using Peredox to measure cytosolic NADH:NAD+ ratios in individual live cells.
NADH; glycolysis; lactate dehydrogenase; sensor calibration; single cell imaging
Several human hepatotropic pathogens including chronic hepatitis C virus (HCV) have narrow species restriction, thus hindering research and therapeutics development against these pathogens. Developing a rodent model that accurately recapitulates hepatotropic pathogens infection, human immune response, chronic hepatitis, and associated immunopathogenesis is essential for research and therapeutics development. Here, we describe the recently developed AFC8 humanized liver- and immune system-mouse model for studying chronic hepatitis C virus and associated human immune response, chronic hepatitis, and liver fibrosis.
Humanized mice; Chronic hepatitis C virus; Liver immunopathogenesis
Dimerization of receptor tyrosine kinases is a well-characterized process. It is imperative for the activation of many receptors, including the epidermal growth factor receptor (EGFR). EGFR has been shown to be regulated by a number of factors, including lipid raft localization. For example, alteration of the lipid raft localization of EGFR has been demonstrated to modify receptor dimerization. This protocol describes an assay to quantify EGFR dimers using BS3 cross-linking. BS3 cross-linking is well suited for this purpose because of its length, water solubility, and membrane impermeability. Although this protocol is written specifically for EGFR, the assay can be extrapolated in order to characterize dimerization of other receptor tyrosine kinases.
Epidermal growth factor receptor; Dimerization; Lipid rafts; BS3; Cross-linking; DHA
Neuropeptides play many important roles in cell-cell signaling and are involved in the control of anxiety, depression, pain, reward pathways, and many other processes that are relevant to psychiatric disorders. Mass spectrometry-based peptidomics techniques can identify the precise forms of peptides that are present in a given tissue. Utilizing this technique, peptides can be identified with any post-translational modifications that may be present and the exact sequence of the peptides can be determined. Unlike radioimmunoassays, which are limited by specific antibodies and often cannot discriminate between different lengths of peptides from the same precursor, peptidomics reveals the precise sequence and allows for the identification of both known and novel peptides. The use of isotopic labels allows for quantitative peptidomics, which results in the ability to compare peptide levels between differently treated samples. These tags can be synthesized in five different isotopic forms, permitting multivariate analysis of up to five different groups of tissue extracts in a single liquid chromatography/mass spectrometry run; this is ideal for measuring changes in neuropeptides in animals subjected to drug treatments, or in comparing animal models of psychiatric disorders.
Peptidomics; proteomics; peptidase; protease
Peptidomics is defined as the analysis of peptides present in a tissue extract, usually using mass spectrometry-based approaches. Unlike radioimmunoassay-based detection techniques, peptidomics measures the precise form of each peptide, including post-translational modifications, and can readily distinguish between longer and shorter forms of the same peptide. Also, peptidomics is not limited to known peptides and can detect hundreds of peptides in a single experiment. Quantitative peptidomics enables comparisons between two or more groups of samples, and is perfect for studies examining the effect of gene knock-outs on tissue levels of peptides. We describe the method for quantitative peptidomics using isotopic labels based on trimethylammonium butyrate, which can be synthesized in five different isotopic forms; this permits multivariate analysis of five different groups of tissue extracts in a single liquid chromatography/mass spectrometry run.
Prohormone convertase; proprotein convertase; carboxypeptidase; peptidomics; proteomics; peptidase; protease
The modular assembly (MA) method of generating engineered zinc finger proteins (ZFPs) was the first practical method for creating custom DNA-binding proteins. As such, MA has enabled a vast exploration of sequence-specific methods and reagents, ushering in the modern era of zinc finger-based applications that are described in this volume. The first zinc finger nuclease to cleave an endogenous site was created using MA, as was the first artificial transcription factor to enter phase II clinical trials. In recent years, other excellent methods have been developed that improved the affinity and specificity of the engineered ZFPs. However, MA is still used widely for many applications. This chapter will describe methods and give guidance for the creation of ZFPs using MA. Such ZFPs might be useful as starting materials to perform other methods described in this volume. Here, we also describe a single-strand annealing recombination assay for the initial testing of zinc finger nucleases.
Zinc finger nuclease; single-strand annealing; homologous recombination; zinc finger protein engineering methods; modular assembly
Small solutes are useful probes of large conformational changes in RNA polymerase (RNAP)-promoter interactions and other biopolymer processes. In general, a large effect of a solute on an equilibrium constant (or rate constant) indicates a large change in water-accessible biopolymer surface area in the corresponding step (or transition state), resulting from conformational changes, interface formation, or both. Here, we describe nitrocellulose filter binding assays from series used to determine the urea dependence of open complex formation and dissociation with Escherichia coli RNAP and λPR promoter DNA. Then, we describe the subsequent data analysis and interpretation of these solute effects.
bacterial RNA polymerase; open complex formation; kinetics; mechanism; solute effects; conformational changes
NMDA receptors (NMDARs) are ionotropic glutamate receptors that are essential for synaptic plasticity, learning and memory. Dysfunction of NMDARs has been implicated in many nervous system disorders; therefore, pharmacological modulation of NMDAR activity has great therapeutic potential. However, given the broad physiological importance of NMDARs, modulating their activity often has detrimental side effects precluding pharmaceutical use of many NMDAR modulators. One approach to possibly improve the therapeutic potential of NMDAR modulators is to identify compounds that modulate subsets of NMDARs. An obvious target for modulating NMDAR subsets are the many NMDAR subtypes produced through different combinations of NMDAR subunits. With seven identified genes that encode NMDAR subunits, there are many neuronal NMDAR subtypes with distinct properties and potentially differential pharmacological sensitivities. Study of NMDAR subtype-specific pharmacology is complicated in neurons, however, because most neurons express at least three NMDAR subtypes. Thus, use of an approach that permits study in isolation of a single receptor subtype is preferred. Additionally, the effects of drugs on agonist-activated responses typically depend on duration of agonist exposure. To evaluate drug effects on synaptic transmission, an approach should be used that allows activation of receptor responses as brief as those observed during synaptic transmission, both in the absence and presence of drug. To address these issues, we designed a fast perfusion system capable of (1) delivering brief (~5 ms) and consistent applications of glutamate to recombinant NMDARs of known subunit composition, and (2) easily and quickly (~5 seconds) changing between glutamate applications in the absence and presence of drug.
NMDA; NMDA receptor subtype; memantine; ketamine; open channel blocker; brief agonist application
DNA assembler enables design and rapid construction of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use the zeaxanthin biosynthetic pathway as an example to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for the construction of the clones, S. cerevisiae transformation, and zeaxanthin production and detection are shown.
DNA assembler; In vivo homologous recombination; Pathway engineering; Synthetic biology; Metabolic engineering; Zeaxanthin biosynthesis
Despite its apparent simplicity, the problem of quantifying the differences between two structures of the same protein or complex is non-trivial and continues evolving. In this chapter, we described several methods routinely used to compare computational models to experimental answers in several modeling assessments. The two major classes of measures, positional distance-based and contact-based, were presented, compared and analyzed.
The most popular measure of the first class, the global RMSD, is shown to be the least representative of the degree of structural similarity because it is dominated by the largest error. Several distance-dependent algorithms designed to attenuate the drawbacks of RMSD are described. Measures of the second class, contact-based, are shown to be more robust and relevant. We also illustrate the importance of using combined measures, utility-based measures, and the role of the distributions derived from the pairs of experimental structures in interpreting the results.
protein structure comparison; modeling; docking; accuracy; assessment; root mean square deviation; atomic contacts; residue contacts; naïve model; Z-score; cumulative distribution function; VLS enrichment
DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated.
DNA assembler; In vivo homologous recombination; Pathway engineering; Synthetic biology; Metabolic engineering; Gene cluster characterization and engineering
Chemical shifts are obtained at the first stage of any protein structural study by NMR spectroscopy. Chemical shifts are known to be impacted by a wide range of structural factors and the artificial neural network based TALOS-N program has been trained to extract backbone and sidechain torsion angles from 1H, 15N and 13C shifts. The program is quite robust, and typically yields backbone torsion angles for more than 90% of the residues, and sidechain χ1 rotamer information for about half of these, in addition to reliably predicting secondary structure. The use of TALOS-N is illustrated for the protein DinI, and torsion angles obtained by TALOS-N analysis from the measured chemical shifts of its backbone and 13Cβ nuclei are compared to those seen in a prior, experimentally determined structure. The program is also particularly useful for generating torsion angle restraints, which then can be used during standard NMR protein structure calculations.
NMR; Chemical shifts; Protein structure; Side-chain conformation; Artificial neural network; Secondary structure; Backbone torsion angle
Multivariate statistical techniques are used extensively in metabolomics studies, ranging from biomarker selection to model building and validation. Two model independent variable selection techniques, principal component analysis and two sample t-tests are discussed in this chapter, as well as classification and regression models and model related variable selection techniques, including partial least squares, logistic regression, support vector machine, and random forest. Model evaluation and validation methods, such as leave-one-out cross-validation, Monte Carlo cross-validation, and receiver operating characteristic analysis, are introduced with an emphasis to avoid over-fitting the data. The advantages and the limitations of the statistical techniques are also discussed in this chapter.
Metabolomics; Mass spectrometry; Multivariate statistics; Classification
The uncoating process of HIV-1 is a poorly understood process, so the development of a reliable assay to study uncoating is critical for moving the field forward. Here we describe an uncoating assay that currently represents the state-of-the-art biochemical procedure for monitoring uncoating and core stability during infection. This assay is based on the biochemical separation of soluble capsid protein from particulate capsid cores and provides information about the fate of the capsid during infection.
HIV-1 core; Uncoating; Capsid; TRIM5α; Sucrose cushion
The endogenous chemotaxis of cells toward sites of tissue injury and/or biomaterial implantation is an important component of the host response. Implanted biomaterials capable of recruiting host stem/progenitor cells to a site of interest may obviate challenges associated with cell transplantation. An assay for the identification and quantification of chemotaxis induced by surgically placed biologic scaffolds composed of extracellular matrix is described herein.
Chemotaxis; Stem cells; Cryptic peptides; Extracellular matrix
High-resolution Fourier-transform mass spectrometry (FTMS) provides important advantages in studies of metabolism because more than half of common intermediary metabolites can be measured in 10 min with minimal pre-detector separation and without ion-dissociation. This allows unprecedented opportunity to study complex metabolic systems, such as mitochondria. Analysis of mouse liver mitochondria using FTMS with liquid chromatography shows that sex and genotypic differences in mitochondrial metabolism can be readily distinguished. Additionally, differences in mitochondrial function are readily measured, and many of the mitochondria-related metabolites are also measurable in plasma. Thus, application of high-resolution mass spectrometry provides an approach for integrated studies of complex metabolic processes of mitochondrial function and dysfunction in disease.
Biostatistics; bioinformatics; environmental chemicals; mass spectrometer; mitochondrial metabolome
Protein prenylation involves the addition of a farnesyl (C15) or geranylgeranyl (C20) isoprenoid moiety onto the C-terminus of approximately 2% of all mammalian proteins. This hydrophobic modification serves to direct membrane association of the protein. Due to the finding that the oncogenic protein Ras is naturally prenylated, several researchers have developed inhibitors of the prenyltransferase enzymes as cancer therapeutics. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation in living cells. Using a combination of flow cytometry and confocal microscopy, we have shown that synthetic fluorescently-labeled prenylated peptides enter a variety of different cell types. Additionally, using capillary electrophoresis we have shown that these peptides can be detected in minute quantities from lysates of cells treated with these peptides. This method will allow for further study of the enzymology of protein prenylation in living cells.
Peptide; lipid modification; post-translational modification; prenylation; farnesyl; cell-penetrating peptide
Gram-negative bacterial outer membrane vesicle production and function have been studied using a variety of quantitative and qualitative methods. These types of analyses can be hampered by the use of impure vesicle preparations. Here we describe a set of techniques that are useful for the quantitative analysis of vesicle production and for preparative yields of highly purified vesicles for studies of vesicle function or composition. Procedures and advice are also included for the purification of vesicles from encapsulated and low-yield strains.
Gram-negative bacteria; outer membrane; vesicle; bleb; Escherichia coli; Pseudomonas aeruginosa; Klebsiella pneumoniae
The rapid expansion of molecular screening libraries in size and complexity in the last decade has outpaced the discovery rate of cost-effective strategies to single out reagents with sought-after cellular activities. In addition to representing high-priority therapeutic targets, intensely studied cell signaling systems encapsulate robust reference points for mapping novel chemical activities given our deep understanding of the molecular mechanisms that support their activity. In this chapter, we describe strategies for using transcriptional reporters of several well-interrogated signal transduction pathways coupled with high-throughput biochemical assays to fingerprint novel compounds for drug target identification agendas.
Small-molecule screening; RNAi; Luciferase assay; Wnt; TP53; Kras; Dot blotting
Peptides and proteins are routinely identified from peptide fragmentation spectra acquired in a mass spectrometer, analyzed by database search engines. The types of fragments that can be formed are known, and it is also well appreciated that certain fragment types are more common or more informative than others. However, most search engines do not use detailed knowledge of peptide fragmentation, but rather consider a limited range of fragments, giving each an equivalent weighting in their scoring system that decides which results are likely to be correct. This chapter will discuss efforts to make use of information about the frequency of observation of different fragment ion types in order to produce more sophisticated and sensitive scoring systems and will demonstrate how these new scoring systems are particularly powerful for analysis of electron capture or electron transfer dissociation data.
Database search engine; collision-induced dissociation; electron transfer dissociation; statistical analysis of fragmentation; charge-state dependent scoring; sequence dependent scoring
Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter we describe methods for isolation of both mtDNA and nuclear DNA (nucDNA), and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material, and availability of specific PCR reagents.
mitochondrial DNA; mtDNA; mtDNA depletion; copy number; QPCR; mitochondrial toxicity; mitochondrial disease
Humanized mice have recently emerged as powerful translational animal models for studying human hematopoiesis, immune interactions, and diseases of the human immune system. Several important advances in the humanized mouse technology have been reported over the last few years, thereby resulting in improved engraftment, high levels of human chimerism, and sustained human hematopoiesis. This chapter describes the detailed procedures for generating various humanized mouse models including hu-PBL, hu-HSC, and BLT models and discusses considerations for choosing the appropriate model system.
Humanized mice; Immunodeficient mice; Integrin; Hematopoietic stem cells; Xenotransplantation; Stem cell transplantation; Human fetal tissue; hu-PBL; hu-HSC; BLT
Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections.
miRNA; Fluorescence in situ hybridization; Formalin-fixed and paraffin-embedded tissues; Molecular diagnostics; Multiplexing; Signal amplification methods