The ubiquitin-proteasome system (UPS) and ubiquitin-related modifiers are not only involved in cellular protein quality control but also in the regulation of many fundamental cellular processes/pathways as well as in their disease-relevant aberrations. Ubiquitin Family Modifiers and Proteasome: Reviews and Protocols presents both novel developments in UPS research and important methods related to the main recent advances in the field of ubiquitin family modifiers. Divided into five convenient sections, this volume focuses on the enzymology and substrate identification of ubiquitin family modifiers, the recognition and chain formation of these modifiers, the analysis of proteasome biogenesis and function, protein quality control, and finally the use of small molecules and strategies to study or manipulate the function of the UPS and of ubiquitin family modifiers, respectively. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Ubiquitin Family Modifiers and Proteasome: Reviews and Protocols will be of great use to investigators and students engaged in both basic and applied research in life sciences.

Protein Structure Prediction, Third Edition expands on previous editions by focusing on software and web servers. With new chapters that provide instructions on how to use a computational method with examples of prediction by the method. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials, step-by-step, readily reproducible protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Protein Structure Prediction, Third Edition provides diverse methods detailing the expansion of the computational protein structure prediction field.

Signal transduction is the fundamental mechanism for regulation of cellular activities by environmental cues and regulatory signals, and is particularly important for plants, whose survival requires proper physiological and developmental responses to the environmental changes. Much progress has been made recently in the plant signal transduction research field thanks to the development of diverse techniques which are covered in Plant Signalling Networks: Methods and Protocols. These include advanced research methods such as proteomics and mass spectrometry methods for studying protein modification, biochemical and cell biological tools for studying protein-protein interactions, genomic techniques for dissecting protein-DNA interaction and transcription networks, and computation methods that integrate molecular network into plant developmental processes. Written in the successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Plant Signalling Networks: Methods and Protocols presents well-honed methodologies for a wide range of research approaches including genetics, proteomics, biochemical, cell biological, and computational approaches, and seeks to serve both professionals and novices with a comprehensive understanding of complex signaling networks in plants.

This book represents proceedings of the 19th American Peptide Symposium. It highlights many of the recent developments in peptide science, with a particular emphasis on how these advances are being applied to basic problems in biology and medicine. Specific topics covered include novel synthetic strategies, peptides in biological signaling, post-translational modifications of peptides and proteins, and peptide quaternary structure in material science and disease.

Now recognized as a reservoir for growth factors and cytokines modulating cell activation status and turnover, proteoglycans have stimulated great amount of interest and research. In Proteoglycans: Methods and Protocols, experts in the field examine issues of basic concepts and up-to-date analysis methods for proteoglycan and glycosaminoglycan (GAG) at the protein and saccharide levels. The volume continues with the multifunctional aspect of proteoglycans, highlighted through three relevant examples of proteoglycans with highly different strucutures: serglycin, aggrecan, and heparin sulphate proteoglycans. The final chapter describes proteoglycan involvement in the pathogenesis of various disorders and their potential therapeutic value in osteo-articular diseases. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Proteoglycans: Methods and Protocols is an ideal guide for scientists attempting to pursue further research in this vital and complex area of study.

The genomes of cellular organisms are organized as double-stranded DNA, a structure that must be unwound to provide DNA replication, recombination, and repair machinery access to genomic information. However, DNA unwinding comes with inherent risks to genome stability. To help mediate these risks, bacterial, archael, and eukaryotic cells have evolved protective ssDNA-binding proteins (SSBs) that bind ssDNA with high affinity and specificity. SSBs also aid genome metabolic processes through direct interactions with key proteins in genome maintenance enzymes. Single-Stranded DNA Binding Proteins: Methods and Protocols assembles methods developed for examining the fundamental properties of SSBs and for exploiting the biochemical functions of SSBs for their use as in vitro and in vivo reagents. Clearly and concisely organized, the volume opens with an introduction to the structures and functions of SSBs, followed protocols for studying SSB/DNA complexes, methods for studying SSB/heterologous protein complexes, protocols for interrogating post-translational modifications of SSBs, and concludes with uses of fluorescently-labeled SSBs for in vitro and in vivo studies of genome maintenance processes. Written in the successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Single-Stranded DNA Binding Proteins: Methods and Protocols provides a rich introduction for investigators who are interested in this fascinating family of DNA-binding proteins.

Thousands of proteins have been identified to be acetylated. Immense research power has been dedicated to experiments to solve the biological implications of each and every protein acetylation. Two particular sites of protein acetylation have been described intensively: the N-terminal methionine residue of a nascent protein and lysine residues within a protein. In Protein Acetylation: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study protein acetylation. These include methods and techniques for identification of protein acetylation, column- and gel electrophoresis-based approaches, computationally prediction, and the biological response to protein acetylation. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protein Acetylation: Methods and Protocols seeks to aid scientists in the further study of the technical aspects involved in understanding protein acetylation.

Progress in functional proteomics has been limited for a long time, partially caused by limitations in assay sensitivity and sample capacity; however, protein microarrays have the ability to overcome these limitations so that a highly parallel analysis of hundreds of proteins in thousands of samples is attainable. In Protein Microarrays: Methods and Protocols, expert researchers in the field present an up-to-date collection of robust strategies in the field of protein microarrays and summarize recent advantages in the field of printing technologies, the development of suitable surface materials, as well as detection and quantification technologies. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Protein Microarrays: Methods and Protocols aims to stimulate the application and further advancement of this powerful technology in labs worldwide.

This book is dedicated to the channels and pores that belong to an eclectic and ubiquitous class of unconventional - perhaps at times strange - pore-forming molecules, which nevertheless play fundamental roles in various organisms. These non-canonical channels may take on various and sometimes complex architectures, such as large beta-barrels or lipid-containing pores. They may originate from bacteria, viruses or intracellular organelles. For some of them, the physiologically relevant substrate may indeed be ions, and for others folded polypeptides. Some are released by cells in a soluble form that has the ability to insert into biological membranes to exert its permeabilizing effect. Many of these unconventional pores have been investigated by electrophysiology, which, by its virtue of focusing on a few or even a single unit, has provided invaluable insight into the mechanisms and structure-function relationships of these remarkable membrane entities. The chapters of this book highlight a representative set of these interesting investigations.

Since the first edition of Protein Nanotechnology Protocols Instruments and Applications the intersection of protein science and nanotechnology has become an exciting frontier in interdisciplinary sciences. The second edition of Protein Nanotechnology Protocols Instruments and Applications expands upon the previous editions with current, detailed chapters that provide examples of proteins which are now being harnessed for a wide range of applications, some more developed than others. This book also delves into engineering proteins and an overview of the sorts of tools that are now readily available to manipulate the structure and function of proteins, both rationally and using methods inspired by evolution. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protein Nanotechnology Protocols Instruments and Applications, Second Edition seeks to provide an overview of this multi-faceted field and a useful guide to those who wish to contribute to it.

The second edition of p53 Protocols expands upon the first edition with progress in p53 research since the publication of the first edition. In p53 Protocols, Second Edition expert researchers in the field detail many of the methods that address the challenging questions of the p53 field. These methods include the identification of the target genes and binding partners of gain of function p53 mutants, methods to determine stress response, autophagy or senescence induced by p53, cell cycle analysis, analysis of different phases of DNA replication modified by p53, and generation of pluripotent stem cells. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, p53 Protocols, Second Edition seeks to aid scientists in the further study into p53 and other tumor suppressors.

In Protein Dynamics: Methods and Protocols, expert researchers in the field detail both experimental and computational methods to interrogate molecular level fluctuations. Chapters detail best-practice recipes covering both experimental and computational techniques, reflecting modern protein research. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protein Dynamics: Methods and Protocols describes the most common and powerful methods used to characterize protein dynamics.

This excellent book covers wide-ranging topics in interdisciplinary microbiology, addressing various research aspects and highlighting advanced discoveries and innovations. It presents the fascinating topic of modern biotechnology, including agricultural microbiology, microalgae biotechnology, bio-energy, bioinformatics and metagenomics, environmental microbiology, enzyme technology and marine biology. It presents the most up-to-date areas of microbiology with an emphasis on shedding light on biotechnological advancements and integrating these interdisciplinary microbiology research topics into other biotechnology sub-disciplines. The book raises awareness of the industrial relevance of microbiology, which is key component of this unique collection. The topics include production of antioxidant-glutathione, enzyme-engineering methods, probiotic microbiology and features of microbial xylanases. It also covers some other remarkable aspects of microbiology, like potential health hazards in recreational water and fullerene nanocomposites, which are vital for biotechnological interventions. This book will be valuable resource for senior undergraduate and graduate students, researchers and other interested professionals or groups working in the interdisciplinary areas of microbiology and biotechnology.

Protein microarrays have been used for a wide variety of important tasks, such as identifying protein-protein interactions, discovering disease biomarkers, identifying DNA-binding specificity by protein variants, and for characterization of the humoral immune response. In Protein Microarray for Disease Analysis: Methods and Protocols, expert researchers provide concise descriptions of the methodologies currently used to fabricate microarrays for the comprehensive analysis of proteins or responses to proteins that can be used to dissect human disease. These methodologies are the toolbox for revolutionizing drug development and cell-level biochemical understanding of human disease processes. Beginning with a section on protein-detecting analytical microarrays, the volume continues with sections covering antigen microarrays for immunoprofiling, protein function microarrays, the validation of candidate targets, proteomic libraries, as well as signal detection strategies and data analysis techniques. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Practical and cutting-edge, Protein Microarray for Disease Analysis: Methods and Protocols serves as a solid framework to aid scientists in understanding how protein microarray technology is presently developing and how it can be applied to transform our analysis of human disease.

This first of two volumes provides a general overview of the genetics, structure, mechanism and regulation of the Ras superfamily proteins and describes in detail the signaling pathways and processes regulated by specific members of this family. The focus of this first volume is on the Rho and Ras subfamily of small G proteins. Renowned scientists provide insights into the biochemistry of the classical and non-classical small G-protein family members, their spatio-temporal regulation, their effectors and their roles in health and disease. Together with Volume 2, this book provides a comprehensive and state-of-the-art work on small G-proteins (GTPases). It is intended for graduates and professors in biochemistry and cell biology already working on small G-proteins (small GTPases), but also offers an extremely valuable resource for those readers who are new to the field.

Display technologies have become a very powerful way of generating therapeutic lead molecules and specific reagents for increasing our understanding of biology; however, despite being first described shortly after phage display, the use of ribosome display and related methods have been much less widespread. Since this is in part due to the complexity of the methods, Ribosome Display and Related Technologies: Methods and Protocols seeks to extend their use by collecting expert contributions describing these detailed protocols. The protocols described range from well-established methods that have been used for a decade to generate high affinity antibodies, which are already in the clinic, to methods that are in their early stages of application such as display of peptides incorporating non-canonical amino acids. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Invaluable and easy to use, Ribosome Display and Related Technologies: Methods and Protocols will be of great benefit to those with general molecular biology or protein engineering experience who wish to select peptides or proteins by display, those with phage display experience who would benefit from the application of ribosome display, as well as those with some ribosome display experience who would like to expand the range of applications to which they are applying the technology.

Proteostasis is central to the development of various human diseases caused due to excessive protein misfolding and the disregulation of the protein quality control system. In this book, respected researchers from many leading institutions contribute their insights on proteostasis maintenance. The coverage mainly focuses on the basics of maintaining proteostasis, the consequences of proteostatic system failure, and how chaperone systems constantly maintain proteostasis. In addition, the book presents in detail different treatment strategies for diseases caused by proteostatic system failure, as well as the inhibition of proteostatic failure using small molecule compounds. It examines advances in the modulation of proteopathies, providing a comprehensive source of key mechanistic insights on these diseases. As such, the book offers a valuable resource for beginners and more experienced investigators alike who are looking for detailed and reliable information on protein homeostasis, the diseases that can develop due to related imbalances and the essential role of molecular and chemical chaperones.

This brief discusses the mechanism of functional expression of a protein or protein complex utilizing the ATP hydrolysis cycle or proton-motive force from a unique point of view focused on the roles of water. A variety of processes are considered such as the unidirectional movement of a linear-motor protein along a filament, insertion of an unfolded protein into a chaperonin and release of the folded protein from it, transport of diverse substrates across the membrane by a transporter, and directed rotation of the central subunit within a rotatory motor protein complex. These topics are discussed in a unified manner within the same theoretical framework. The author argues that water plays imperative roles in the functional expression of these molecular machines. A pivotal factor is the entropic force or potential originating from the translational displacement of water molecules coexisting with the molecular machines in the entire system.

This volume focuses on various aspects of interleukin-27 (IL-27), especially its potential for clinical applications. The authors discuss the downstream signaling from the IL-27 receptor and its molecular targets in immune cells including Th1, Th2, Th17, Treg, Tr1, Tfh, B cells, DCs and macrophages. The inhibition of Th17 cells by IL-27 is vital for the maintenance of the feto-maternal tolerance and the prevention of lupus, multiple sclerosis, autoimmune uveitis, immune thrombocytopenia and atherosclerosis. However, the same inhibitory capabilities compromise the immune response to bacterial pathogens, and IL-27 is a pathogenic factor in sepsis and tuberculosis. Also covered are the conflicting reports on the role of IL-27 in rheumatoid arthritis, the effect of IL-27 on epithelia, which seems to play a role in asthma, psoriasis and inflammatory bowel diseases, and the direct cytotoxic and anti-vascular effects of IL-27, which make it a promising agent for the treatment of cancer. Accordingly, this volume will be of interest to researchers and clinicians alike.

NMR spectroscopy has proven to be a powerful technique to study the structure and dynamics of biological macromolecules. Fundamentals of Protein NMR Spectroscopy is a comprehensive textbook that guides the reader from a basic understanding of the phenomenological properties of magnetic resonance to the application and interpretation of modern multi-dimensional NMR experiments on 15N/13C-labeled proteins. Beginning with elementary quantum mechanics, a set of practical rules is presented and used to describe many commonly employed multi-dimensional, multi-nuclear NMR pulse sequences. A modular analysis of NMR pulse sequence building blocks also provides a basis for understanding and developing novel pulse programs. This text not only covers topics from chemical shift assignment to protein structure refinement, as well as the analysis of protein dynamics and chemical kinetics, but also provides a practical guide to many aspects of modern spectrometer hardware, sample preparation, experimental set-up, and data processing. End of chapter exercises are included to emphasize important concepts. Fundamentals of Protein NMR Spectroscopy not only offer students a systematic, in-depth, understanding of modern NMR spectroscopy and its application to biomolecular systems, but will also be a useful reference for the experienced investigator.

The study of carbonic anhydrase has spanned multiple generations of scientists. Carbonic anhydrase was first discovered in 1932 by Meldrum and Roughton. Inhibition by sulfanilamide was shown in 1940 by Mann and Keilin. Even Hans Krebs contributed to early studies with a paper in 1948 showing the relationship of 25 different sulfonamides to CA inhibition. It was he who pointed out the importance of both the charged and uncharged character of these compounds for physiological experiments. The field of study that focuses on carbonic anhydrase (CA) has exploded in recent years with the identification of new families and isoforms. The CAs are metalloenzymes which are comprised of 5 structurally different families: the alpha, beta, gamma, and delta, and epsilon classes. The alpha class is found primarily in animals with several isoforms associated with human disease. The beta CAs are expressed primarily in plants and are the most divergent. The gamma CAs are the most ancient. These are structurally related to the beta CAs, but have a mechanism more similar to the alpha CAs. The delta CAs are found in marine algae and diflagellates. The epsilon class is found in prokaryotes in which it is part of the carboxysome shell perhaps supplying RuBisCO with CO2 for carbon fixation. With the excitement surrounding the discovery of disease-related CAs, scientists have redoubled their efforts to better understand structure-function relationships, to design high affinity, isotype-specific inhibitors, and to delineate signaling systems that play regulatory roles over expression and activity. We have designed the book to cover basic information of mechanism, structure, and function of the CA families. The authors included in this book bring to light the newest data with regard to the role of CA in physiology and pathology, across phylums, and in unique environmental niches.

G protein-coupled receptors (GPCRs) are heptahelical transmembrane receptors that convert extra-cellular stimuli into intra-cellular signaling, and ultimately into biological responses. Since GPCRs are natural targets for approximately 40% of all modern medicines, it is not surprising that they have been the subject of intense research. Notwithstanding the amount of data generated over the years, discovering ligands of these receptors with optimal therapeutic properties is not straightforward and has certainly been hampered for years by the lack of high-resolution structural information about these receptors. Luckily, there has been a steady increase of high-resolution crystal structures of these receptors since 2007, and this information, integrated with dynamic inferences from computational and experimental methods, holds great potential for the discovery of new, improved drugs. This book, which provides, for the first time, state-of-the-art views on modeling and simulation of GPCRs, is divided into 4 parts. In the first part, the impact of currently available GPCR crystal structures on structural modeling is discussed extensively as are critical insights from simulations in the second part of the book. The third part reports recent progress in rational ligand discovery and mathematical modeling, whereas the fourth part provides an overview of bioinformatics tools and resources that are available for GPCRs.

This book addresses the differentiation control of skeletal muscle in different locations of the vertebrate body Particular attention is paid to novel regulatory molecules and signals as well as the heterogeneity of origin that have revealed a developmental overlap between skeletal and cardiac muscle. Different functional muscle groups are the product of the evolution of the vertebrate classes, making a phylogenetic comparison worthwhile for understanding the role of muscle stem cells and precursors in myogenesis. New insights into the hierarchy of transcription factors, particularly in the context of these different muscle groups have been gained from detailed investigations of the spatio-temporal and regulatory relationships derived from mouse and zebrafish genetics and avian microsurgery. Importantly, epigenetic mechanisms that have surfaced recently, in particular the role of MyomiRs, are also surveyed. With an eye to the human patient, encouraging results have been generated that identify parallels between embryonic myogenesis and regenerating myofibers due to common regulatory molecules. On the other hand, both processes differ considerably in quality and complexity of the processes employed. Interestingly, the heterogeneity in embryonic sources from which skeletal muscle groups in the vertebrate including the human body take origin is paralleled by differences in their susceptibility to particular muscle dystrophies as well as by the characteristics of the satellite cells involved in regeneration. The progress that has been made in the field of muscle stem cell biology, with special focus on the satellite cells, is outlined in this book by experts in the field. The authors review recent insights of the heterogeneous nature of these satellite cells regarding their gene signatures and regeneration potential. Furthermore, an improved understanding of muscle stem cells seems only possible when we study the impact of the cell environment on efficient stem cell replacement therapies for muscular dystrophies, putting embryological findings from different vertebrate classes and stem cell approaches into context.

This book covers the latest developments in the physical biology of proteins and peptides. Key insights into microscopic and macroscopic approaches to describe biologically relevant macromolecules and their interactions are provided. This book also covers a wide range of tools, including theoretical methods as statistical mechanics, normal mode analysis, kinetic theory and stochastic processes, and all-atom and coarse-grained molecular dynamics simulations. New experimental techniques are also discussed, particularly related to amiloidogenic peptides and their mutations. This is an excellent book for molecular biologists, physicists, computational scientists, and chemists. It covers cutting-edge research in this exciting, interdisciplinary research field. This book also: Discusses the latest developments in the physical biology of proteins, peptides and enzymes covering theoretical, computational, and experimental approaches Broadens readers' understanding on the r ole of intra- and inter-molecular interactions as a fundamental cornerstone of macroscopic biological properties of macromolecules Provides a wide and useful perspective on different aspects of the physics, biology, and chemistry of proteins and peptides suitable for interdisciplinary research.

In this thesis, the author outlines the development of new monolithic columns and isotope dimethyl labeling strategies and their applications in high-performance proteome analyses. Though different types of monolithic columns have been widely developed for chromatography and electrophoresis separation, their application in proteomics for complex peptide mixtures separation is still a challenge. The author discusses the preparation of new monolithic columns and optimization of chromatography separation capability to improve coverage and accuracy of proteome analysis. Further, the author describes a novel online multidimensional chromatography system combined with automated online isotope labeling, which significantly improves the throughput, sensitivity and accuracy of quantitative proteomics. In addition to the development of new technologies, the author investigates the proteome and phosphoproteome expression changes of clinical hepatocellular carcinoma tissues and the hippocampi of mice with Alzheimer’s disease. The work in this thesis has led to several publications in high-profile journals in the fields of analytical chemistry and proteome research.