Showing papers in "Methods in Enzymology in 2010"

Real-time DNA sequencing from single polymerase molecules.

Abstract: Pacific Biosciences has developed a method for real-time sequencing of single DNA molecules (Eid et al., 2009), with intrinsic sequencing rates of several bases per second and read lengths into the kilobase range. Conceptually, this sequencing approach is based on eavesdropping on the activity of DNA polymerase carrying out template-directed DNA polymerization. Performed in a highly parallel operational mode, sequential base additions catalyzed by each polymerase are detected with terminal phosphate-linked, fluorescence-labeled nucleotides. This chapter will first outline the principle of this single-molecule, real-time (SMRT) DNA sequencing method, followed by descriptions of its underlying components and typical sequencing run conditions. Two examples are provided which illustrate that, in addition to the DNA sequence, the dynamics of DNA polymerization from each enzyme molecules is directly accessible: the determination of base-specific kinetic parameters from single-molecule sequencing reads, and the characterization of DNA synthesis rate heterogeneities.

Accurate Single-Molecule FRET Studies Using Multiparameter Fluorescence Detection

Abstract: In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures In particular, Forster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in a population of molecules After a short introduction, the data analysis procedure is described in detail together with some experimental considerations The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure determination based on FRET A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when determining FRET-related distances with high accuracy Furthermore, the broad time resolution (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomolecule of interest explores its structural energy landscape

Synthetic genetic array (SGA) analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Abstract: A genetic interaction occurs when the combination of two mutations leads to an unexpected phenotype. Screens for synthetic genetic interactions have been used extensively to identify genes whose products are functionally related. In particular, synthetic lethal genetic interactions often identify genes that buffer one another or impinge on the same essential pathway. For the yeast Saccharomyces cerevisiae, we developed a method termed synthetic genetic array (SGA) analysis, which offers an efficient approach for the systematic construction of double mutants and enables a global analysis of synthetic genetic interactions. In a typical SGA screen, a query mutation is crossed to an ordered array of ~5000 viable gene deletion mutants (representing ~80% of all yeast genes) such that meiotic progeny harboring both mutations can be scored for fitness defects. This approach can be extended to all ~6000 genes through the use of yeast arrays containing mutants carrying conditional or hypomorphic alleles of essential genes. Estimating the fitness for the two single mutants and their corresponding double mutant enables a quantitative measurement of genetic interactions, distinguishing negative (synthetic lethal) and positive (within pathway and suppression) interactions. The profile of genetic interactions represents a rich phenotypic signature for each gene and clustering genetic interaction profiles group genes into functionally relevant pathways and complexes. This array-based approach automates yeast genetic analysis in general and can be easily adapted for a number of different genetic screens or combined with high-content screening systems to quantify the activity of specific reporters in genome-wide sets of single or more complex multiple mutant backgrounds. Comparison of genetic and chemical-genetic interaction profiles offers the potential to link bioactive compounds to their targets. Finally, we also developed an SGA system for the fission yeast Schizosaccharomyces pombe, providing another model system for comparative analysis of genetic networks and testing the conservation of genetic networks over millions of years of evolution.

Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes.

Abstract: Measurements of gene expression within single cells have revealed startling variability otherwise hidden in bulk measurements. Here, we present an in situ hybridization method capable of detecting individual mRNA molecules, thus permitting the accurate quantification and localization of mRNA within fixed sample. Our in situ protocol involves probing the target mRNA using a series of singly labeled oligonucleotide probes. This method is simple to implement and is applicable to a variety of biological samples. We also discuss some aspects of image processing required for analyzing the resulting data.

Genome-Wide Translational Profiling by Ribosome Footprinting

Abstract: We present a detailed protocol for ribosome profiling, an approach that we developed to make comprehensive and quantitative measurements of translation in yeast. In this technique, ribosome positions are determined from their nuclease footprint on their mRNA template and the footprints are quantified by deep sequencing. Ribosome profiling has already enabled highly reproducible measurements of translational control. Because this technique reports on the exact position of ribosomes, it also revealed the presence of ribosomes on upstream open reading frames and demonstrated that ribosome density was higher near the beginning of protein-coding genes. Here, we describe nuclease digestion conditions that produce uniform ~28 nucleotide (nt) protected fragments of mRNA templates that indicate the exact position of translating ribosomes. We also give a protocol for converting these RNA fragments into a DNA library that can be sequenced using the Illumina Genome Analyzer. Unbiased conversion of anonymous, small RNAs into a sequencing library is challenging, and we discuss standards that played a key role in optimizing library generation. Finally, we discuss how deep sequencing data can be used to quantify gene expression at the level of translation.

GraFix: stabilization of fragile macromolecular complexes for single particle cryo-EM.

Abstract: Here, we review the GraFix (Gradient Fixation) method to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy (cryo-EM). During GraFix, macromolecules undergo a weak, intramolecular chemical cross-linking while being purified by density gradient ultracentrifugation. GraFix-stabilized particles can be used directly for negative-stain cryo-EM or, after a brief buffer-exchange step, for unstained cryo-EM. This highly reproducible method has proved to dramatically reduce problems in heterogeneity due to particle dissociation during EM grid preparation. Additionally, there is often an appreciable increase in particles binding to the carbon support film. This and the fact that binding times can be drastically increased, with no apparent disruption of the native structures of the macromolecules, makes GraFix a method of choice when preparing low-abundance complexes for cryo-EM. The higher sample quality following GraFix purification is evident when examining raw images, which usually present a low background of fragmented particles, good particle dispersion, and high-contrast, well-defined particles. Setting up the GraFix method is straightforward, and the resulting improvement in sample homogeneity has been beneficial in successfully obtaining the 3D structures of numerous macromolecular complexes by cryo-EM in the past few years.

Analysis of complex single-molecule fret time trajectories

Abstract: Single-molecule methods have given researchers the ability to investigate the structural dynamics of biomolecules at unprecedented resolution and sensitivity. One of the preferred methods of studying single biomolecules is single-molecule fluorescence resonance energy transfer (smFRET). The popularity of smFRET stems from its ability to report on dynamic, either intra- or intermolecular interactions in real-time. For example, smFRET has been successfully used to characterize the role of dynamics in functional RNAs and their protein complexes, including ribozymes, the ribosome, and more recently the spliceosome. Being able to reliably extract quantitative kinetic and conformational parameters from smFRET experiments is crucial for the interpretation of their results. The need for efficient, unbiased analysis routines becomes more evident as the systems studied become more complex. In this chapter, we focus on the practical utility of statistical algorithms, particularly hidden Markov models, to aid in the objective quantification of complex smFRET trajectories with three or more discrete states, and to extract kinetic information from the trajectories. Additionally, we present a method for systematically eliminating transitions associated with uncorrelated fluorophore behavior that may occur due to dye anisotropy and quenching effects. We also highlight the importance of data condensation through the use of various transition density plots to fully understand the underlying conformational dynamics and kinetic behavior of the biological macromolecule of interest under varying conditions. Finally, the application of these techniques to studies of pre-mRNA conformational changes during eukaryotic splicing is discussed.

Molecular Genetics of Schizosaccharomyces pombe

Abstract: In this chapter we present basic protocols for the use of Schizosaccharomyces pombe, commonly known as fission yeast, in molecular biology and genetics research. Fission yeast is an increasingly popular model organism for the study of biological pathways because of its genetic tractability and as a model for metazoan biology. It provides an alternative and complimentary approach to Saccharomyces cerevisiae for addressing questions of cell biology, physiology, genetics, and genomics/proteomics. We include details and considerations for growing fission yeast, information on crosses and genetics, gene targeting and transformation, cell synchrony and analysis, and molecular biology protocols.

High-quality binary interactome mapping.

Abstract: Physical interactions mediated by proteins are critical for most cellular functions and altogether form a complex macromolecular "interactome" network. Systematic mapping of protein-protein, protein-DNA, protein-RNA, and protein-metabolite interactions at the scale of the whole proteome can advance understanding of interactome networks with applications ranging from single protein functional characterization to discoveries on local and global systems properties. Since the early efforts at mapping protein-protein interactome networks a decade ago, the field has progressed rapidly giving rise to a growing number of interactome maps produced using high-throughput implementations of either binary protein-protein interaction assays or co-complex protein association methods. Although high-throughput methods are often thought to necessarily produce lower quality information than low-throughput experiments, we have recently demonstrated that proteome-scale interactome datasets can be produced with equal or superior quality than that observed in literature-curated datasets derived from large numbers of small-scale experiments. In addition to performing all experimental steps thoroughly and including all necessary controls and quality standards, careful verification of all interacting pairs and validation tests using independent, orthogonal assays are crucial to ensure the release of interactome maps of the highest possible quality. This chapter describes a high-quality, high-throughput binary protein-protein interactome mapping pipeline that includes these features.

Microbe-associated molecular patterns in innate immunity: Extraction and chemical analysis of gram-negative bacterial lipopolysaccharides.

Abstract: Bacterial lipopolysaccharides (LPSs) are the major component of the outer membrane of Gram-negative bacteria. They have a structural role since they contribute to the cellular rigidity by increasing the strength of cell wall and mediating contacts with the external environment that can induce structural changes to allow life in different conditions. Furthermore, the low permeability of the outer membrane acts as a barrier to protect bacteria from host-derived antimicrobial compounds. They also have a very important role in the elicitation of the animal and plant host innate immunity since they are microbe-associated molecular patterns, namely, they are glycoconjugates produced only by Gram-negative bacteria and are recognized as a molecular hallmark of invading microbes. LPSs are amphiphilic macromolecules generally comprising three defined regions distinguished by their genetics, structures, and function: the lipid A, the core oligosaccharide and a polysaccharide portion, the O-chain. In some Gram-negative bacteria, LPS can terminate with the core portion to form rough-type LPS (R-LPS, LOS). In this chapter, we will describe the isolation of both kinds of LPSs and their full chemical analysis, pivotal operations in the complete description of the primary structure of such important glycoconjugates.

Analysis of single nucleic acid molecules with protein nanopores

Abstract: We describe the methods used in our laboratory for the analysis of single nucleic acid molecules with protein nanopores. The technical section is preceded by a review of the variety of experiments that can be done with protein nanopores. The end goal of much of this work is single-molecule DNA sequencing, although sequencing is not discussed explicitly here. The technical section covers the equipment required for nucleic acid analysis, the preparation and storage of the necessary materials, and aspects of signal processing and data analysis.

Resolution measures in molecular electron microscopy

Abstract: Resolution measures in molecular electron microscopy provide means to evaluate quality of macromolecular structures computed from sets of their two-dimensional (2D) line projections. When the amount of detail in the computed density map is low there are no external standards by which the resolution of the result can be judged. Instead, resolution measures in molecular electron microscopy evaluate consistency of the results in reciprocal space and present it as a one-dimensional (1D) function of the modulus of spatial frequency. Here we provide description of standard resolution measures commonly used in electron microscopy. We point out that the organizing principle is the relationship between these measures and the spectral signal-to-noise ratio (SSNR) of the computed density map. Within this framework it becomes straightforward to describe the connection between the outcome of resolution evaluations and the quality of electron microscopy maps, in particular, the optimum filtration, in the Wiener sense, of the computed map. We also provide a discussion of practical difficulties of evaluation of resolution in electron microscopy, particularly in terms of its sensitivity to data processing operations used during structure determination process in single particle analysis and in electron tomography (ET).

Use of Glycan Microarrays to Explore Specificity of Glycan-Binding Proteins

Abstract: Microarrays of defined glycans represent a high throughput approach to determining the specificity of lectins, or more generally glycan-binding proteins (GBPs). The utility of a glycan microarray is directly related to the number and variety of the glycans available on the printed surface for interrogation by GBPs. The Consortium for Functional Glycomics (CFG), funded by the National Institute of General Medical Sciences (NIGMS), has generated a glycan microarray available to the public as an investigator-driven resource, where hundreds of GBPs have been analyzed. Here we describe the methods generally used by the CFG to prepare glycan arrays and interrogate them with GBPs. We also describe our new approach to normalizing glycan microarray data derived from concentration-dependent analyses of GBP binding, and the application of this approach with the plant lectin Sambucus nigra agglutinin (SNA-I) and human galectin-8. The use of glycan microarrays with this approach readily generates a prediction of the glycan determinants required for high affinity binding by a GBP.

Single-Molecule Fluorescence Studies of Intrinsically Disordered Proteins

Abstract: Intrinsically disordered proteins (IDPs) (also referred to as natively unfolded proteins) play critical roles in a variety of cellular processes such as transcription and translation and also are linked to several human diseases. Biophysical studies of IDPs present unusual experimental challenges due in part to their broad conformational heterogeneity and potentially complex binding-induced folding behavior. By minimizing the averaging over an ensemble (which is typical of most conventional experiments), single-molecule fluorescence (SMF) techniques have recently begun to add advanced capabilities for structural studies to the experimental arsenal of IDP investigators. Here, we briefly discuss a few common SMF methods that are particularly useful for IDP studies, including SMF resonance energy transfer and fluorescence correlation spectroscopy, along with site-specific protein-labeling methods that are essential for application of these methods to IDPs. We then present an overview of a few studies in this area, highlighting how SMF methods are being used to gain valuable information about two amyloidogenic IDPs, the Parkinson's disease-linked α-synuclein and the NM domain of the yeast prion protein Sup 35. SMF experiments provided new information about the proteins' rapidly fluctuating IDP forms, and the complex α-synuclein folding behavior upon its binding to lipid and membrane mimics. We anticipate that SMF and single-molecule methods, in general, will find broad application for structural and mechanistic studies of a wide variety of IDPs, both of their disordered conformations, and their ordered ensembles relevant for function and disease.

DNA curtains for high-throughput single-molecule optical imaging.

Abstract: Single-molecule approaches provide a valuable tool in the arsenal of the modern biologist, and new discoveries continue to be made possible through the use of these state-of-the-art technologies. However, it can be inherently difficult to obtain statistically relevant data from experimental approaches specifically designed to probe individual reactions. This problem is compounded with more complex biochemical reactions, heterogeneous systems, and/or reactions requiring the use of long DNA substrates. Here we give an overview of a technology developed in our laboratory, which relies upon simple micro- or nanofabricated structures in combination with “bio-friendly” lipid bilayers, to align thousands of long DNA molecules into defined patterns on the surface of a microfluidic sample chamber. We call these “DNA curtains,” and we have developed several different versions varying in complexity and DNA substrate configuration, which are designed to meet different experimental needs. This novel approach to single-molecule imaging provides a powerful experimental platform that offers the potential for concurrent observation of hundreds or even thousands of protein–DNA interactions in real time.

Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins.

Abstract: Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines.

Radiation Damage in Electron Cryomicroscopy

Abstract: In an electron microscope, the electron beam used to determine the structures of biological tissues, cells, and molecules destroys the specimen as the image is acquired. This destruction occurs before a statistically well-defined image can be obtained and is consequently the fundamental limit to resolution in biological electron cryomicroscopy (cryo-EM). Damage from the destructive interaction of electrons with frozen-hydrated specimens occurs in three stages: primary damage, as electrons ionize the sample, break bonds, and produce secondary electrons and free radicals; secondary damage, as the secondary electrons and free radicals migrate through the specimen and cause further chemical reactions; and tertiary damage, as hydrogen gas is evolved within the sample, causing gross morphological changes to the specimen. The deleterious effects of radiation are minimized in cryo-EM by limiting the exposure of the specimen to incident electrons and cooling the sample to reduce secondary damage. This review emphasizes practical considerations for minimizing radiation damage, including measurement of electron exposure, estimation of absorbed doses of energy, selection of microscope voltage and specimen temperature, and selection of electron exposure to optimize images.

Mapping cell fate and function using recombinase-based intersectional strategies

Abstract: Cell types are typically defined by expression of a unique combination of genes, rather than a single gene. Intersectional methods therefore become crucial to selectively access these cells for higher resolution fate mapping and functional manipulations. Here, we discuss one such intersectional method. Two recombinase systems (Cre/loxP and Flp/FRT) work together to remove a double STOP cassette and thereby activate expression of a target transgene solely in cells defined by a particular pairwise combination of driver genes. Depending on the nature of the target transgene, this strategy can be used to deliver cell-lineage tracers, sensors, and/or effector molecules to highly selective cell types in vivo. In this chapter, we discuss concepts, reagents, and methods underlying this intersectional approach and encourage consideration of various intersectional and binary methods for accessing uniquely defined cell subsets in the mouse.

Reconstruction of helical filaments and tubes.

Abstract: While Fourier-Bessel methods gave rise to the first three-dimensional reconstruction of an object from electron microscopic images, and these methods have dominated three-dimensional reconstruction of helical filaments and tubes for 30 years, single-particle approaches to helical reconstruction have emerged within the past 10 years that are now the main method being used The Iterative Helical Real Space Reconstruction (IHRSR) approach has been the main methodology, and it surmounts many of the problems posed by real polymers that are flexible, display less than crystalline order, or are weakly scattering The main difficulty in applying this method, or even Fourier-Bessel methods, is in determining the approximate helical symmetry This chapter focuses on some of the intrinsic ambiguities that are present when trying to determine the helical symmetry from power spectra of images and argues that complementary techniques or some form of prior knowledge about the subunit may be needed to have confidence in the solution that is found

The Ashwell-Morell receptor.

Abstract: The Ashwell-Morell receptor (AMR) of hepatocytes, originally termed the hepatic asialoglycoprotein receptor, was the first cellular receptor to be identified and isolated and the first lectin to be detected in mammals. It is one of the multiple lectins of the C-type lectin family involved in recognition, binding, and clearance of asialoglycoproteins. We recently identified endogenous ligands of the AMR as desialylated prothrombotic components, including platelets and von Willebrand Factor [Ellies L. G., Ditto D., Levy G. G., Wahrenbrock M., Ginsburg D., Varki A., Le D. T., and Marth J. D. (2002). Sialyltransferase ST3Gal-IV operates as a dominant modifier of hemostasis by concealing asialoglycoprotein receptor ligands. Proc. Natl. Acad. Sci. USA 99: pp. 10042-10047; Grewal, P. K. Uchiyama, S., Ditto, D., Varki, N., Le, D. T., Nizet, V., Marth, J. D. (2008). The Ashwell receptor mitigates the lethal coagulopathy of sepsis. Nat. Medicine 14, pp. 648-655]. Among these components, clearance by the liver's AMR is enhanced by exposure of terminal galactose on the glycan chains. A physiological role for engaging the AMR in rapid clearance was identified as mitigating disseminating intravascular coagulopathy in sepsis to promote survival. This chapter overviews the endogenous ligands of the AMR as components of the coagulatory system, describes clearance mechanisms of the liver, and details hematology and coagulation assays used in mouse coagulation studies.

Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins.

Abstract: Sulfenic acids, formed as transient intermediates during the reaction of cysteine residues with peroxides, play significant roles in enzyme catalysis and regulation, and are also involved in the redox regulation of transcription factors and other signaling proteins. Therefore, interest in the identification of protein sulfenic acids has grown substantially in the past few years. Dimedone, which specifically traps sulfenic acids, has provided the basis for the synthesis of a novel group of compounds that derivatize 1,3-cyclohexadione, a dimedone analogue, with reporter tags such as biotin for affinity capture and fluorescent labels for visual detection. These reagents allow identification of the cysteine sites and proteins that are sensitive to oxidation and permit identification of the cellular conditions under which such oxidations occur. We have shown that these compounds are reactive and specific toward sulfenic acids and that the labeled proteins can be detected at high sensitivity using gel analysis or mass spectrometry. Here, we further characterize these reagents, showing that the DCP-Bio1 incorporation rates into three sulfenic acid containing proteins, papaya papain, Escherichia coli fRMsr, and the Salmonella typhimurium peroxiredoxin AhpC, are significantly different and, in the case of fRMsr, are unaffected by changes in buffer pH from 5.5 and 8.0. We also provide protocols to label protein sulfenic acids in cellular proteins, either by in situ labeling of intact cells or by labeling at the time of lysis. We show that the addition of alkylating reagents and catalase to the lysis buffer is critical in preventing the formation of sulfenic acid subsequent to cell lysis. Data presented herein also indicate that the need to standardize, as much as possible, the protein and reagent concentrations during labeling. Finally, we introduce several new test or control proteins that can be used to evaluate labeling procedures and efficiencies.

Fundamentals of three-dimensional reconstruction from projections.

Abstract: Three-dimensional (3D) reconstruction of an object mass density from the set of its 2D line projections lies at a core of both single-particle reconstruction technique and electron tomography. Both techniques utilize electron microscope to collect a set of projections of either multiple objects representing in principle the same macromolecular complex in an isolated form, or a subcellular structure isolated in situ. Therefore, the goal of macromolecular electron microscopy is to invert the projection transformation to recover the distribution of the mass density of the original object. The problem is interesting in that in its discrete form it is ill-posed and not invertible. Various algorithms have been proposed to cope with the practical difficulties of this inversion problem and their differ widely in terms of their robustness with respect to noise in the data, completeness of the collected projection dataset, errors in projections orientation parameters, abilities to efficiently handle large datasets, and other obstacles typically encountered in molecular electron microscopy. Here, we review the theoretical foundations of 3D reconstruction from line projections followed by an overview of reconstruction algorithms routinely used in practice of electron microscopy.

Plunge Freezing for Electron Cryomicroscopy

Abstract: Aqueous biological samples must be “preserved” (stabilized) before they can be placed in the high vacuum of an electron microscope. Among the various approaches that have been developed, plunge freezing maintains the sample in the most native state and is therefore the method of choice when possible. Plunge freezing for standard electron cryomicroscopy applications proceeds by spreading the sample into a thin film across an EM grid and then rapidly submerging it in a cryogen (usually liquid ethane), but success depends critically on the properties of the grid and sample, the production of a uniformly thin film, the temperature and nature of the cryogen, and the plunging conditions. This chapter reviews plunge-freezing principles, techniques, instrumentation, common problems, and safety considerations.

Redox state of human serum albumin in terms of cysteine-34 in health and disease.

Abstract: Albumin, the main protein in plasma, is prone to different mechanisms of oxidative modification since extracellular fluids contain only small amounts of antioxidant enzymes. The redox state of cysteine-34 of human albumin defines three fractions which allow to monitor albumin oxidation: mercaptalbumin with a free thiol group, nonmercaptalbumin1 containing a disulfide, and nonmercaptalbumin2 with a sulfinic or sulfonic acid group. These fractions can be separated by HPLC and detected with UV or fluorescence detection. The method is very rugged and only simple sample preparation is needed. It has been used to demonstrate albumin oxidation during exercise, aging, and pathologies like diabetes, liver disease, or renal disease. Problems may arise when high endogenous concentrations of bilirubin or certain drugs are present. The redox state of albumin shows high variability and is hence a valuable tool for the investigation of reversible and irreversible modification of plasma protein.

Quantitative genetic interaction mapping using the E-MAP approach.

Abstract: Genetic interactions represent the degree to which the presence of one mutation modulates the phenotype of a second mutation. In recent years, approaches for measuring genetic interactions systematically and quantitatively have proven to be effective tools for unbiased characterization of gene function and have provided valuable data for analyses of evolution. Here, we present protocols for systematic measurement of genetic interactions with respect to organismal growth rate for two yeast species.

Combining Optical Tweezers, Single-Molecule Fluorescence Microscopy, and Microfluidics for Studies of DNA–Protein Interactions

Abstract: The technically challenging field of single-molecule biophysics has established itself in the last decade by granting access to detailed information about the fate of individual biomolecules, unattainable in traditional biochemical assays. The appeal of single-molecule methods lies in the directness of the information obtained from individual biomolecules. Technological improvements in single-molecule methods have made it possible to combine optical tweezers, fluorescence microscopy, and microfluidic flow systems. Such a combination of techniques has opened new possibilities to study complex biochemical reactions on the single-molecule level. In this chapter, we provide general considerations for the development of a combined optical trapping, fluorescence microscopy, and microfluidics instrument, along with methods to solve technical issues that are critical for designing successful experiments. Finally, we present several experiments to illustrate the power of this combination of techniques.

A bird's eye view tracking slow nanometer-scale movements of single molecular nano-assemblies.

Abstract: Recent improvements in methods of single-particle fluorescence tracking have permitted detailed studies of molecular motion on the nanometer scale. In a quest to introduce these tools to the burgeoning field of DNA nanotechnology, we have exploited fluorescence imaging with one-nanometer accuracy (FIONA) and single-molecule high-resolution colocalization (SHREC) to monitor the diffusive behavior of synthetic molecular walkers, dubbed "spiders," at the single-molecule level. Here we discuss the imaging methods used, results from tracking individual spiders on pseudo-one-dimensional surfaces, and some of the unique experimental challenges presented by the low velocities (approximately 3 nm/min) of these nanowalkers. These experiments demonstrate the promise of fluorescent particle tracking as a tool for the detailed characterization of synthetic molecular nanosystems at the single-molecule level.

Classification of structural heterogeneity by maximum-likelihood methods.

Abstract: With the advent of computationally feasible approaches to maximum-likelihood (ML) image processing for cryo-electron microscopy, these methods have proven particularly useful in the classification of structurally heterogeneous single-particle data. A growing number of experimental studies have applied these algorithms to study macromolecular complexes with a wide range of structural variability, including nonstoichiometric complex formation, large conformational changes, and combinations of both. This chapter aims to share the practical experience that has been gained from the application of these novel approaches. Current insights on how to prepare the data and how to perform two- or three-dimensional classifications are discussed together with the aspects related to high-performance computing. Thereby, this chapter will hopefully be of practical use for those microscopists wishing to apply ML methods in their own investigations.

Changing paradigms in thiology from antioxidant defense toward redox regulation.

Abstract: The history of free radical biochemistry is briefly reviewed in respect to major trend shifts from the focus on radiation damage toward enzymology of radical production and removal and ultimately the role of radicals, hydroperoxides, and related fast reacting compounds in metabolic regulation. Selected aspects of the chemistry of radicals and hydroperoxides, the enzymology of peroxidases, and the biochemistry of adaptive responses and regulatory phenomena are compiled and discussed under the perspective of how the fragments of knowledge can be merged to biologically meaningful concepts of regulation. It is concluded that (i) not radicals but H(2)O(2), hydroperoxides, and peroxynitrite are the best candidates for oxidant signals, (ii) peroxidases of the GPx and Prx family or functionally equivalent proteins have the chance to specifically sense hydroperoxides and to transduce the oxidant signal, (iii) redox signaling proceeds via reactions known from thiol peroxidase and redoxin chemistry, (iv) proximal targets are proteins that are modified at SH groups, and (v) redoxins are documented signal transducers but also used as terminators. The importance of kinetics for forward signaling and for sensitivity modulation by competition is emphasized and ways to restore resting conditions are discussed. Research needs to validate emerging concepts are outlined.

An introduction to maximum-likelihood methods in cryo-EM.

Abstract: The maximum-likelihood method provides a powerful approach to many problems in cryo-electron microscopy (cryo-EM) image processing. This contribution aims to provide an accessible introduction to the underlying theory and reviews existing applications in the field. In addition, current developments to reduce computational costs and to improve the statistical description of cryo-EM images are discussed. Combined with the increasing power of modern computers and yet unexplored possibilities provided by theory, these developments are expected to turn the statistical approach into an essential image-processing tool for the electron microscopist.