Accurate measurements of the thermodynamic stability of folded membrane proteins require methods for monitoring their conformation that are free of experimental artifacts. For tryptophan fluorescence emission experiments with membrane proteins folded into liposomes, there are two significant sources of artifacts: the first is light scattering by the liposomes; the second is the nonlinear relationship of some tryptophan spectral parameters with changes in protein conformation. Both of these sources of error can interfere with the method of determining the reversible equilibrium thermodynamic stability of proteins using titrations of chemical denaturants. Here, we present methods to manage light scattering by liposomes for tryptophan emission experiments and to properly monitor tryptophan spectra as a function of protein conformation. Our methods are tailored to the titrations of membrane proteins using common chemical denaturants. One of our recommendations is to collect and analyze the right-angle light scattering peak that occurs around the excitation wave- length in a fluorescence experiment. Another recommendation is to use only those tryptophan spectral parameters that are linearly proportional to the protein conformational population. We show that other commonly used spectral commonly used parameters lead to errors in protein stability measurements.
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 canbeusedtoevaluate labeling procedures and efficiencies.
Luminescence reporters have been used successfully in studies of circadian rhythms. Real-time measurements of circadian variations in gene expression were made in living cells, cultured tissues, and whole organisms. Because this technique is relatively easy and continuous noninvasive measurement from tissue cultures allows for a drastic reduction in the number of experimental animals, we believe this method will become a common technique for studying circadian rhythms. Using a multichannel recording apparatus, it may also become a powerful tool for the discovery of new drugs. In the past, measurements were done using hand-made apparatuses or by modifying commercially available equipment. We, along with other investigators, have developed user-friendly equipment for performing circadian rhythms experiments, and these systems are now available commercially. This article describes the use of luminescence reporters in circadian research and provides detailed methods used in these experiments. One of our goals in this article is to reduce experimental variability in different laboratories by proposing standard protocols.
Chimeric mice are versatile model systems for the study of mammalian circadian biology. In chimeras, genetically different cells are combined within single animals, making them useful for assessing how normal cells interact with genetically altered cells in intact biological systems. In particular, the primary circadian pacemaker in the suprachiasmatic nucleus is amenable to analysis using series of chimeras that incorporate cells carrying mutations in circadian genes. The study of chimeras carrying circadian mutations can contribute to a better understanding of the function of the altered genes and of the fundamental physiology of circadian timing. Chimera analysis is a valuable approach for studying network properties in complex, integrated biological systems like that, which controls circadian behavior in mammals.
Liposomes are composed of lipid bilayer membranes that encapsulate an aqueous volume. A major challenge in the development of liposomes for drug delivery is the control of size and size distribution. In conventional methods, lipids are spontaneously assembled into heterogeneous bilayers in a bulk phase. Additional processing by extrusion or sonication is required to obtain liposomes with small size and a narrow size distribution. Microfluidics is an emerging technology for liposome synthesis, because it enables precise control of the lipid hydration process. Here, we describe a number of microfluidic methods that have been reported to produce micro/nanosized liposomes with narrower size distribution in a reproducible manner, focusing on the use of continuous-flow microfluidics. The advantages of liposome formation using the microfluidic approach over traditional bulk-mixing approaches are discussed.
A universal goal in studying the structures of macromolecules and macromolecular complexes by means of electron cryo-microscopy (cryo-TEM) and three-dimensional (3D) image reconstruction is the derivation of a reliable atomic or pseudoatomic model. Such a model provides the foundation for exploring in detail the mechanisms by which biomolecules function. Though a variety of highly ordered, symmetric specimens such as 2D crystals, helices, and icosahedral virus capsids have been studied by these methods at near-atomic resolution, until recently, numerous challenges have made it difficult to achieve sub-nanometer resolution with large (≥~500 Å), asymmetric molecules such as the tailed bacteriophages.
After briefly reviewing some of the history behind the development of asymmetric virus reconstructions, we use recent structural studies of the prolate phage φ29 as an example to illustrate the step-by-step procedures used to compute an asymmetric reconstruction at sub-nanometer resolution. In contrast to methods that have been employed to study other asymmetric complexes, we demonstrate how symmetries in the head and tail components of the phage can be exploited to obtain the structure of the entire phage in an expedited, stepwise process. Prospects for future enhancements to the procedures currently employed are noted in the concluding section.
The enhanced permeability and retention (EPR) effect has been a key rationale for the development of nanoscale carriers to solid tumors. As a consequence of EPR, nanotherapeutics are expected to improve drug and detection probe delivery, have less adverse effects than conventional chemotherapy, and thus result in improved detection and treatment of tumors. Physiological barriers posed by the abnormal tumor microenvironment, however, can hinder the homogeneous delivery of nanomedicine in amounts sufficient to eradicate cancer. To effectively enhance the therapeutic outcome of cancer patients by nanotherapeutics, we have to find ways to overcome these barriers. One possibility is to exploit the abnormal tumor microenvironment for selective and improved delivery of therapeutic agents to tumors. Recently, we proposed a multistage nanoparticle delivery system as a potential means to enable uniform delivery throughout the tumor and improve the efficacy of anticancer therapy. Here, we describe the synthesis of a novel multistage nanoparticle formulation that shrinks in size once it enters the tumor interstitial space to optimize the delivery to tumors as well as within tumors. Finally, we provide detailed experimental methods for the characterization of such nanoparticles.
Forward genetic approaches (phenotype to gene) are powerful methods to identify mouse circadian clock components. The success of these approaches, however, is highly dependent on the quality of the phenotype— specifically, the ability to measure circadian rhythms in individual mice. This article outlines the factors necessary to measure mouse circadian rhythms, including choice of mouse strain, facilities and equipment design and construction, experimental design, high-throughput methods, and finally methods for data analysis.
Liposomal dry powder formulations (DPFs) have proven their superiority over conventional DPFs due to favorably improved pharmacokinetics and pharmacodynamics of entrapped drugs, and thus, reduced local and systemic toxicities. Nanoliposomal DPFs (NLDPFs) provide stable, high aerosolization efficiency to deep lung, prolonged drug release, slow systemic dilution, and avoid macrophage uptake of encapsulated drug by carrier-based delivery of nano-range liposomes. This chapter describes methods of preparation of nanoliposomes (NLs) and NLDPFs, using various techniques, and their characterization with respect to size distribution, flow behavior, in vitro drug release profile, lung deposition, cellular uptake and cytotoxicity, and in vivo pharmacokinetics and pharmacodynamics. Some examples have been detailed for better understanding of the methods of preparation and evaluation of NLDPFs by investigators.
Logic-embedded vectors (LEVs) have been introduced as a means to overcome sequential, biological barriers that prevent particle-based drug delivery systems from reaching their targets. In this chapter, we address the challenge of fabricating and optimizing LEVs to reach non-endosomal targets. We describe the general preparation, characterization, and cellular association of porous silicon-based LEVs. A specific example of LEV fabrication from start to finish, along with optimization and troubleshooting information, is presented to serve as a template for future designs.
Ghrelin O-acyltransferase (GOAT) is responsible for catalyzing the attachment of the eight-carbon fatty acid octanoyl to the Ser3 side chain of the peptide ghrelin to generate the active form of this metabolic hormone. As such, GOAT is viewed as a potential therapeutic target for the treatment of obesity and diabetes mellitus. Here, we review recent progress in the development of cell and in vitro assays to measure GOAT action and the identification of several synthetic GOAT inhibitors. In particular, we discuss the design, synthesis, and characterization of the bisubstrate analog GO-CoA-Tat and its ability to modulate weight and blood glucose in mice. We also highlight current challenges and future research directions in our biomedical understanding of this fascinating ghrelin processing enzyme.
Antibody responses are initiated by the binding of antigens to clonally distributed cell surface B cell receptors (BCRs) that trigger signaling cascades resulting in B cell activation. Using conventional biochemical approaches, the components of the downstream BCR signaling pathways have been described in considerable detail. However, far less is known about the early molecular events by which the binding of antigens to the BCRs initiates BCR signaling. With the recent advent of high-resolution, high-speed, live-cell and single-molecule imaging technologies, these events are just beginning to be elucidated. Understanding the molecular mechanisms underlying the initiation of BCR signaling may provide new targets for therapeutics to block dysregulated BCR signaling in systemic autoimmune diseases and in B cell tumors and to aid in the design of protein subunit vaccines. In this chapter we describe the general procedures for using these new imaging techniques to investigate the early events in the initiation of BCR signaling.
This article describes the methods and techniques used to produce mutagenized mice to conduct high-throughput forward genetic screens for circadian rhythm mutants in the mouse. In particular, we outline methods to safely prepare and administer the chemical mutagen N-nitrosoN-ethylurea (ENU) to mice. We also discuss the importance of selecting mouse strain and outline breeding strategies, logistics, and throughput to produce these mutant mice. Finally, we discuss the breeding strategies that we use to confirm mutation heritability.
A critical challenge of the postgenomic era is to understand how genes are differentially regulated. Genetic and genomic approaches have been used successfully to assign genes to distinct regulatory networks in both prokaryotes and eukaryotes. However, little is known about what determines the differential expression of genes within a particular network, even when it involves a single transcription factor. The fact that coregulated genes may be differentially expressed suggests that subtle differences in the shared cis-acting regulatory elements are likely to be significant. This chapter describes a method, termed gene promoter scan (GPS), that discriminates among coregulated promoters by simultaneously considering a variety of cis-acting regulatory features. Application of this method to the PhoP/PhoQ two-component regulatory system of Escherichia coli and Salmonella enterica uncovered novel members of the PhoP regulon, as well as regulatory interactions that had not been discovered using previous approaches. The predictions made by GPS were validated experimentally to establish that the PhoP protein uses multiple mechanisms to control gene transcription and is a central element in a highly connected network.
Sampling alternative conformations is key to understanding how proteins work and engineering them for new functions. However, accurately characterizing and modeling protein conformational ensembles remains experimentally and computationally challenging. These challenges must be met before protein conformational heterogeneity can be exploited in protein engineering and design. Here, as a stepping stone, we describe methods to detect alternative conformations in proteins and strategies to model these near-native conformational changes based on backrub-type Monte Carlo moves in Rosetta. We illustrate how Rosetta simulations that apply backrub moves improve modeling of point mutant side chain conformations, native side chain conformational heterogeneity, functional conformational changes, tolerated sequence space, protein interaction specificity, and amino acid co-variation across protein-protein interfaces. We include relevant Rosetta command lines and RosettaScripts to encourage the application of these types of simulations to other systems. Our work highlights that critical scoring and sampling improvements will be necessary to approximate conformational landscapes. Challenges for the future development of these methods include modeling conformational changes that propagate away from designed mutation sites and modulating backbone flexibility to predictively design functionally important conformational heterogeneity.
Protein design; protein dynamics; conformational heterogeneity; conformational sampling; alternative conformations; Rosetta; Ringer; Backrub
Proteoglycans represent a structurally heterogeneous family of proteins that typically undergo extensive posttranslational modification with sulfated sugar chains. Although historically believed to affect signaling pathways exclusively as growth factor coreceptors, proteoglycans are now understood to initiate and modulate signal transduction cascades independently of other receptors.
From within the extracellular matrix, proteoglycans are able to shield protein growth factors from circulating proteases and establish gradients that guide cell migration. Extracellular proteoglycans are also critical in the maintenance of growth factor stores and are thus instrumental in modulating paracrine signaling.
At the cell membrane, proteoglycans stabilize ligand–receptor interactions, creating potentiated ternary signaling complexes that regulate cell proliferation, endocytosis, migration, growth factor sensitivity, and matrix adhesion. In some cases, proteoglycans are able to independently activate various signaling cascades, attenuate the signaling of growth factors, or orchestrate multimeric intracellular signaling complexes. Signaling between cells is also modulated by proteoglycan activity at the cell membrane, as exemplified by the proteoglycan requirement for effective synaptogenesis between neurons.
Finally, proteoglycans are able to regulate signaling from intracellular compartments, particularly in the context of storage granule formation and maintenance. These proteoglycans are also major determinants of exocytic vesicle fate and other vesicular trafficking pathways.
In contrast to the mechanisms underlying classical ligand-receptor signaling, proteoglycan signaling is frequently characterized by ligand promiscuity and low-affinity binding; likewise, these events commonly do not exhibit the same degree of reliance on intermolecular structure or charge configurations as other ligand-receptor interactions. Such unique features often defy conventional mechanisms of signal transduction, and present unique challenges to the study of their indispensable roles within cell signaling networks.
Histone posttranslational modifications (PTMs) play a pivotal role in regulating the dynamics and function of chromatin. Supported by an increasing body of literature, histone PTMs such as methylation and acetylation function together in the context of a “histone code,” which is read, or interpreted, by effector proteins that then drive a functional output in chromatin (e.g., gene transcription). A growing number of domains that interact with histones and/or their PTMs have been identified. While significant advances have been made in our understanding of how these domains interact with histones, a wide number of putative histone-binding motifs have yet to be characterized, and undoubtedly, novel domains will continue to be discovered. In this chapter, we provide a detailed method for the construction of combinatorially modified histone peptides, microarray fabrication using these peptides, and methods to characterize the interaction of effector proteins, antibodies, and the substrate specificity of histone-modifying enzymes. We discuss these methods in the context of other available technologies and provide a user-friendly approach to enable the exploration of histone–protein–enzyme interactions and function.
Mating pheromone receptors of the yeast Saccharomyces cerevisiae are useful models for the study of G protein-coupled receptors. The mating pheromone receptors, Ste2 and Ste3, are not essential for viability so they can be readily targeted for analysis by a variety of genetic approaches. This chapter will describe methods for identification of two kinds of mutants that have been very informative about the mechanisms of receptor signaling: constitutively-active mutants and dominant-negative mutants. Interestingly, these distinct types of mutants have revealed complementary information. Constitutive signaling is caused by mutations that are thought to weaken interactions between the seven transmembrane domains, whereas the dominant-negative mutants apparently stabilize contacts between transmembrane domains and lock receptors in the off conformation. In support of these conclusions, certain combinations of constitutively-active and dominant-negative mutants restore nearly normal signaling properties.
Tuberculosis is one of the world's most prevalent infectious diseases. The causative agent, M. tuberculosis, asymptomatically infects more than 30% of the world population and causes 8 million cases of active disease and 2 million deaths annually. Its pathogenic success stems from its ability to block phagolysosome biogenesis and subsequent destruction in the host macrophages. Recently, our laboratory has uncovered autophagy as a new means of overcoming this block and promoting the killing of mycobacteria. Here we describe the methods to study autophagy during M. tuberculosis infection of macrophages. The described assays can be used to investigate and identify factors important for autophagic elimination of mycobacteria that could potentially provide new therapeutic targets to defeat this disease.
Accurate energy functions are critical to macromolecular modeling and design. We describe new tools for identifying inaccuracies in energy functions and guiding their improvement, and illustrate the application of these tools to improvement of the Rosetta energy function. The feature analysis tool identifies discrepancies between structures deposited in the PDB and low energy structures generated by Rosetta; these likely arise from inaccuracies in the energy function. The optE tool optimizes the weights on the different components of the energy function by maximizing the recapitulation of a wide range of experimental observations. We use the tools to examine three proposed modifications to the Rosetta energy function: improving the unfolded state energy model (reference energies), using bicubic spline interpolation to generate knowledge based torisonal potentials, and incorporating the recently developed Dunbrack 2010 rotamer library (Shapovalov and Dunbrack, 2011).
Rosetta; energy function; scientific benchmarking; parameter estimation; decoy discrimination
To avoid the challenges of crystallization and the size limitations of NMR, it has long been hoped that single-particle cryo-electron microscopy (cryo-EM) would eventually yield atomically interpretable reconstructions. For the most favorable class of specimens (large icosahedral viruses), one of the key obstacles is curvature of the Ewald sphere, which leads to a breakdown of the Projection Theorem used by conventional three-dimensional (3D) reconstruction programs. Here, we review the basic problem and our implementation of the “paraboloid” reconstruction method, which overcomes the limitation by averaging information from images recorded from different points of view.
Protein phosphorylation is a major form of posttranslational modification critical to cell signaling that also occurs in mitochondrial proteome. Yet, only very limited studies have been performed to characterize mitochondrial-targeted protein kinases or phosphatases. Recently, we identified a novel member of PP2C family (PP2Cm) that is a resident mitochondrial protein phosphatase which plays an important role in normal development and cell survival. In this chapter, we will describe the methods applied in the identification of PP2Cm as a resident mitochondrial protein phosphatase based on sequence analysis and biochemical characterization. We will also provide experimental protocols used to establish the intracellular localization of PP2Cm, to achieve loss and gain function of PP2Cm in cultured cells and intact tissue, and to assess the impact of PP2Cm deficiency on cell death, mitochondria oxidative phosphorylation and permeability transition pore opening.
Mining for novel natural compounds is of eminent importance owing to the continuous need for new pharmaceuticals. Filamentous fungi are historically known to harbor the genetic capacity for an arsenal of natural compounds, both beneficial and detrimental to humans. The majority of these metabolites are still cryptic or silent under standard laboratory culture conditions. Mining for these cryptic natural products can be an excellent source for identifying new compound classes. Capitalizing on the current knowledge on how secondary metabolite gene clusters are regulated has allowed the research community to unlock many hidden fungal treasures, as described in this chapter.
The endoplasmic reticulum (ER) functions to properly fold and process secreted and transmembrane proteins. Environmental and genetic factors that disrupt ER function cause an accumulation of misfolded and unfolded proteins in the ER lumen, a condition termed ER stress. ER stress activates a signaling network called the Unfolded Protein Response (UPR) to alleviate this stress and restore ER homeostasis, promoting cell survival and adaptation. However, under unresolvable ER stress conditions, the UPR promotes apoptosis. Here we discuss the current methods to measure ER stress levels, UPR activation, and subsequent pathways in mammalian cells. These methods will assist us in understanding the UPR and its contribution to ER stress related-disorders such as diabetes and neurodegeneration.
Visual pigment proteins belong to the superfamily of G protein-coupled receptors and are the light-sensitive molecules in rod and cone photoreceptor cells. The protein moiety is known as opsin and the ligand in the dark is 11-cis retinal, which serves as both the photon detector and an inverse agonist. While much is known about properties of the rod pigment rhodopsin, much less is understood about cone visual pigments. Being able to identify ligands that effect opsins give an insight into structure–activity relationships. The action of some ligands indicates that there are differences between not only rod and cone opsins but also among the different classes of cone opsins. Furthermore, inverse agonists of cone opsins may have potential therapeutic uses under conditions when the native 11-cis retinal ligand is absent. A method for determining the effects of ligands on rod and cone opsin activity is described.