The first volume in this Methods Molecular Biology series, Proteins (1984), concentrated on basic techniques for the analysis and purification of peptides and proteins. As the series developed, more specialized volumes on proteins were introduced, such as those on Immunochemical Protocols (vol. 10), Practical Protein Chro- tography (vol. 11), Analysis Glycoprotein Biomedicine (vol. 14), Protein-DNA Interactions (vol. 30), Biomembrane Protocols (vols. 19 and 27), Analyses and Methods (vol. 17), and Optical Spectroscopy, Microscopy, and Macroscopic Techniques (vol. 22). Further specialist volumes on peptides, monoclonal antibodies, immunoassays, ELISA, protein engineering, protein stability, mass spectrometry of proteins, automated sequence analysis, and protein NMR are currently in preparation. Since it is now a decade since the initial volume was published, it seems an especially appropriate moment to extensively reorganize, update, and revise the earlier volume. In an attempt to be more c- prehensive in our coverage, this current volume, Basic Protein and Peptide Protocols, is totally committed to basic analytical methods; a planned companion volume will later concentrate on preparative techniques. Those analytical techniques requiring expensive speci- ized instrumentation, such as NMR, mass spectrometry, X-ray cr- tallography, spectroscopy, and automated sequence analysis, are not described here, but in the appropriate specialized volumes listed above.

A Safety Considerations Many techniques described here involve a number of hazards, such as high electrical current and voltage, radioactivity and highly toxic chemicals. It is absolutely essential that the instructions of equipment manufacturers be followed, and that particular attention be paid to the local and federal safety regulations. B Introduction The expression of prokaryotic and eukaryotic genes has been shown most often to be regulated at the level of mRNA synthesis. Thanks to the rapid development of methods for dissecting DNA sequences, cis-acting regulatory elements such as promoters and enhancers have been recognised. More recently, the widely expressed intuition that discrete sequences within these elements constitute binding sites for sequence-specific binding proteins has been confirmed, especially through the use of "footprinting" assays (for examples, Galas and Schmitz, 1978). This and similar assays have already resulted in the recognition, isolation and analysis of DNA-bind ing proteins for several genes. Excellent reviews exist of the structural studies on these transcription regulatory proteins and related DNA elements (for example, Glover, 1989 and Johnson and McKnight, 1989), to which the reader is referred for detailed information. To set the scene for applications of the techniques described in this volume, only the barest outline of previous studies is presented here. Protein-DNA interactions are dependent on very specific tertiary configurations of the binding protein which allow the closest contact with the DNA helix.

There is increasing evidence for the clinicial value of the apo lipoprotein measurements. Besides cholesterol in plasma and li poprotein fractions, which is currently used as an indicator of cardiovascular risk, the measurement of the AI and B apolipopro teins can provide additional information about the patients' clinical status. Several studies show that apo B is higher and apo AI is lower in patients with angiographically documented coronary heart dis ease than in symptomatic patients without coronary heart disease. Moreover, discriminant analysis indicated that the concentration of Apo AI and B in plasma are better discriminators than lipo protein cholesterol for identifying patients with coronary heart disease. In some studies the apo Bjapo AI ratio appears to be a more powerful predictor than individual lipoproteins. In a recent study carried out in men, apolipoproteins AI and B were better correlated with the severity of cardiovascular disease than HDL and LDL cholesterol. The predictive power of apolipoproteins could however not be demonstrated in all studies and the value of apolipoprotein measurements in the field of clinical chemistry is still controversial. This is probably due to discrepancies between the results of various studies, arising from differences in the type of immunoassays, the lack of universal reference materials, differ ences between study protocols, variations in the selection of patients and in the grading and interpretation of coronary lesions."

One of the phylogenetically oldest hormones, somatostatin is a regulatory peptide with remarkable characteristics. It is a nonclassical neurotransmitter discovered less than 20 years ago both in the central nervous system and in the gastroenteropancreatic system. It regulates the secretion of both pituitary and digestive hormones, it ensures nutrient homeostasis and it has therapeutic uses. This volume deals withall these aspects.

Peptide therapy has become a key strategy in innovative drug development, however, one of the potential barriers for the development of novel peptide drugs in the clinic is their deficiencies in clearly defined chemistry, manufacturing and controls (CMC) strategy from clinical development to commercialization. CMC can often become a rate-limiting step due to lack of knowledge and lack of a formal policy or guidelines on CMC for peptide-based drugs. Regulators use a risk-based approach, reviewing applications on a case-by-case basis. Peptide Therapeutics: Strategy and Tactics for Chemistry, Manufacturing, and Controls covers efficient manufacturing of peptide drug substances, a review of the process for submitting applications to the regulatory authority for drug approval, a holistic approach for quality attributes and quality control from a regulatory perspective, emerging analytical tools for the characterisation of impurities, and the assessment of stability. This book is an essential reference work for students and researchers, in both academia and industry, with an interest in learning about CMC, and facilitating development and manufacture of peptide-based drugs.

In recent years there has been a tremendous increase in our understanding of the functioning of the cell at the molecular level. This has been achieved in the main by the invention and development of new methodology, parti- larly in that area generally referred to as "'genetic en- neering." While this revolution has been taking place in the field of nucleic acids research, the protein chemist has at the same time developed fresh methodology to keep pace with the requirements of present day molecular bi- ogy. Today's molecular biologist can no longer be content with being an expert in one particular area alone. He/she needs to be equally competent in the laboratory at h- dling DNA, RNA, and proteins, moving from one area to another as required by the problem he/she is trying to solve. Although many of the new techniques in molecular biology are relatively easy to master, it is often difficult for a researcher to obtain all the relevant information nec- sary for setting up and successfully applying a new te- nique. Information is of course available in the research l- erature, but this often lacks the depth of description that the new user requires. This requirement for in-depth pr- tical details has become apparent by the considerable - mand for places on our Molecular Biology Workshops held at Hatfield each summer.

The elucidation of the ultrastructure, cytochemistry and reactivity of cutaneous recep- tors is of particular importance in the unsolved biological problem of sensory transduc- tion. Topographically and with regard to evolution, cutaneous receptors are the most widespread primary sense organs. Because of their simple structure they are particular- ly useful in the study of the functional morphology of receptor end-organs. Research into receptor structures in classic neuromorphology revealed magnificent patterns and pictures of receptors, showing their great diversity and their presence in all human and animal systems. Since the end of the sixties, ultrastructural studies have led to the discovery of a large amount of new information. The findings concerning the cellular organization of the inner core (pease and Quilliam, 1957), axon terminals (Cauna and Ross, 1960) and capsule cells (Polacek and Mazanek, 1966) of encapsulated receptors have opened up new possibilities for a revaluation of receptor cell units and their relationships.

This snapshot volume is designed to provide a smooth entry into the field of protein folding. Presented in a concise manner, each section introduces key concepts while providing a brief overview of the relevant literature. Outlook subsections will pinpoint specific aspects related to emerging methodologies, concepts and trends.

This book provides a comprehensive overview of the basic principles, concepts, techniques and latest advances in the field of biomembranes and membrane-associated processes. With new emerging technologies and bioinformatics tools, this is a promising area for future study and research. The book discusses the composition, fluidity and dynamic nature of phospholipid bilayers, which vary with cell/organelle type and function. It describes the various types of transport proteins that facilitate the transport of polar and nonpolar molecules across the membrane actively or passively via ion-channels or through porins. It also explores the many cellular functions membranes participate in: (1) energy transduction, which includes the electron transport chain in inner membrane of mitochondria and bacterial cytoplasmic membrane and photosynthetic electron transport in thylakoid membranes in chloroplast and photosynthetic bacterial membranes; (2) cell-cell communication involving various signal transduction pathways triggered by activated membrane receptors; (3) cell-cell interactions involving various types of adhesion and receptor proteins; (4) nerve transmission involving opening and closing of voltage gated ionic channels; and (5) intracellular transport involving the processes of endocytosis, exocytosis, vesicular transport of solutes between intracellular compartments, membrane fusion and membrane biogenesis.

This monograph is neither a historical outline of the development of the concepts of protein biosynthesis and the structure and functions of the ribosomes, nor an exhaustive survey of the literature on these questions. The monograph is based upon an analysis of the modern trends in this field. The purpose of the monograph was to formulate more or less generalized representations of the structure and" function of the ribosome, as we envision it at the present day. It may be that this attempt is premature for a number of reasons, and the concepts outlined here will very soon be revised. Nonetheless, despite this risk, we believe it to be advisable to undertake this attempt for the following reasons: firstly, the undertaken analysis could aid in the comprehension of the substantial mass of extremely scattered experimental data on the ribosomes presently available; secondly, in any event, even if most of the concepts outlined rapidly become obselete, they can still serve as a stimulus for a whole series of experiments; and thirdly, we hope that some of the concepts outlined will still remain essentially correct and relatively stable. In view of the aforementioned, we should make the following reservations. First of all, we made no attempt to cite all the literature on the problems discussed, but considered it sufficient to illustrate the various premises with one or several sample references.

The handling and analysis of data generated by proteomics investigations represent a challenge for computer scientists, biostatisticians, and biologists to develop tools for storing, retrieving, visualizing, and analyzing genomic data. Informatics in Proteomics examines the ongoing advances in the application of bioinformatics to proteomics research and analysis. Through computer simulations, scientists can determine more about how diseases affect cells, predict how various drug interventions would work, and ultimately use proteins as therapeutic targets. This book first addresses the infrastructure needed for public protein databases. It discusses information management systems and user interfaces for storage, retrieval, and visualization of the data as well as issues surrounding data standardization and integration of protein sequences recorded in the last two decades. The authors subsequently examine the application of statistical and bioinformatic tools to data analysis, data presentation, and data mining. They discuss the implementation of algorithms, statistical methods, and computer applications that facilitate pattern recognition and biomarker discovery by integrating data from multiple sources. This book offers a well-rounded resource of informatic approaches to data storage, retrieval, and protein analysis as well as application-specific bioinformatic tools that can be used in disease detection, diagnosis, and treatment. Informatics in Proteomics captures the current state-of-the-art and provides a valuable foundation for future directions.

This text offers in-depth perspectives on every aspect of protein structure identification, assessment, characterization, and utilization, for a clear understanding of the diversity of protein shapes, variations in protein function, and structure-based drug design. The authors cover numerous high-throughput technologies as well as computational methods to study protein structures and residues. A valuable reference, this book reflects current trends in the effort to solve new structures arising from genome initiatives, details methods to detect and identify errors in the prediction of protein structural models, and outlines challenges in the conversion of routine processes into high-throughput platforms.

While the genomic revolution has quickly led to the deposit of more than 30,000 structures in the protein data bank (PDB), less than one percent of those contributions represent membrane proteins despite the fact that membrane proteins constitute some 20 percent of all proteins. This discrepancy becomes significantly troublesome when it is coupled with the fact that 60 percent of current drugs are based on targeting this group of proteins, a trend that does not seem likely to reverse. Structural Genomics on Membrane Proteins provides an excellent overview on novel research in bioinformatics and modeling on membranes, as well as the latest technological developments being employed in expression, purification, and crystallography to obtain high-resolution structures on membrane proteins. This cutting-edge work also explains the difficulties facing researchers-both technical and ethical-that have slowed the process. Structural Genomics on Membrane Proteins provides researchers with an unprecedented look at the novel technologies that will ultimately allow them to conquer the last frontier in structural biology, leading to accelerated breakthroughs in drug discovery.

Beginning from centuries of anecdotal descriptions of cell death, such as those on the development of the midwife toad in 1842 by Carl Vogt, to modern-day investigations of cell death as a biological discipline, it has become accepted that cell death in multicellular organisms is a normal part of life.This book provides a comprehensive view of cell death, from its mechanisms of initiation and execution, to its implication in human disease and therapy.

Physiological cell death plays critical roles in almost all aspects of biology, and the book details its roles in lymphocyte homeostasis, neuronal function, metabolism, and the DNA damage response.When physiological cell death goes awry, diseases can arise, and cancer is presented as a central paradigm for the consequences of derangements in the interplay between cell survival and cell death.At the same time, the potential promise of targeted therapies aimed at interdicting cell death machineries are also discussed extensively.The molecular mechanisms that underlie apoptotic cell death are illustrated from the perspectives of both the intrinsic, mitochondrial apoptotic pathway and the extrinsic, death receptor pathway.Key players in these pathways, such as the Bcl2 family proteins, cytochrome c, Apaf-1, caspases, death receptor adapter proteins, and inhibitor of apoptosis proteins, are presented from both functional and structural angles. Until only a few years ago, programmed cell death has been considered essentially synonymous with apoptosis.However, we now know that programmed cell death can also take other forms such as necrosis or necroptosis, and to this end, the mechanisms that underlie programmed necrosis in development and host defense are illustrated.The past twenty plus years have seen an incredible growth of research in cell death, with one breakthrough after another, and the legacy still goes on with constant new surprises and findings.Long live cell death "

This is the first serious attempt to synthesize all that became known of glutathione over the last three decades. The book contains an update of glutathione biosynthesis with special emphasis on its regulation in adaptive stress responses. Other chapters review glutathione transport systems and glutathione peroxidases and their differences in substrate specificities and localization. Further contributions center on the diversified roles of different glutathione-S-transferases and the roles of nitrosoglutathione and glutaredoxins - a subfamily of redoxins. The book closes with discussions of the analogous or homologous thiol metabolism in pathogens and the potential suitability of involved enzymes as drug targets. Key selling features: Summarizing the way glutathione is involved in stress responses Compiling the multiple ways glutathione affects inflammatory responses Disclosing how glutathione dampens programmed cell death such as ferroptosis Exploring the enigma of how enzymes accelerate glutathione-dependent processes Discussing how detoxification and redox regulation is mediated by glutathionylation Reviewing the ways glutaredoxins catalyze protein disulfide reduction Highlighting the medical impact of glutathione-related metabolic pathways Illustrating the role thiol metabolism of pathogens might play in drug discovery

Sets the stage for the design and application of new protein cages

Featuring contributions from a team of international experts in the coordination chemistry of biological systems, this book enables readers to understand and take advantage of the fascinating internal molecular environment of protein cages. With the aid of modern organic and polymer techniques, the authors explain step by step how to design and construct a variety of protein cages. Moreover, the authors describe current applications of protein cages, setting the foundation for the development of new applications in biology, nanotechnology, synthetic chemistry, and other disciplines.

Based on a thorough review of the literature as well as the authors' own laboratory experience, "Coordination Chemistry in Protein Cages"Sets forth the principles of coordination reactions in natural protein cagesDetails the fundamental design of coordination sites of small artificial metalloproteins as the basis for protein cage designDescribes the supramolecular design and assembly of protein cages for or by metal coordinationExamines the latest applications of protein cages in biology and nanotechnologyDescribes the principles of coordination chemistry that govern self-assembly of synthetic cage-like molecules

Chapters are filled with detailed figures to help readers understand the complex structure, design, and application of protein cages. Extensive references at the end of each chapter serve as a gateway to important original research studies and reviews in the field.

With its detailed review of basic principles, design, and applications, "Coordination Chemistry in Protein Cages" is recommended for investigators working in biological inorganic chemistry, biological organic chemistry, and nanoscience.

Vibrational Spectroscopy in Protein Research offers a thorough discussion of vibrational spectroscopy in protein research, providing researchers with clear, practical guidance on methods employed, areas of application, and modes of analysis. With chapter contributions from international leaders in the field, the book addresses basic principles of vibrational spectroscopy in protein research, instrumentation and technologies available, sampling methods, quantitative analysis, origin of group frequencies, and qualitative interpretation. In addition to discussing vibrational spectroscopy for the analysis of purified proteins, chapter authors also examine its use in studying complex protein systems, including protein aggregates, fibrous proteins, membrane proteins and protein assemblies. Emphasis throughout the book is placed on applications in human tissue, cell development, and disease analysis, with chapters dedicated to studies of molecular changes that occur during disease progression, as well as identifying changes in tissues and cells in disease studies.

"Introducing Proteomics" gives a concise and coherent overview of every aspect of current proteomics technology, which is a rapidly developing field that is having a major impact within the life and medical sciences.

This student-friendly book, based on a successful course developed by the author, provides its readers with sufficient theoretical background to be able to plan, prepare, and analyze a proteomics study.

The text covers the following: Separation TechnologiesAnalysis of Peptides/Proteins by Mass SpectrometryStrategies in Proteomics

This contemporary text also includes numerous examples and explanations for why particular strategies are better than others for certain applications. In addition, "Introducing Proteomics" includes extensive references and a list of relevant proteomics information sources; essential for any student.

This no-nonsense approach to the subject tells students exactly what they need to know, leaving out unnecessary information. The student companion site enhances learning and provides answers to the end of chapter problems.

"I think this book will be a popular and valuable resource for students and newcomers to the field who would like to have an overview and initial understanding of what proteomics is about. The contents are well organized and address the major issues."
--Professor Walter Kolch, Director, Systems Biology Ireland & Conway Institute, University College Dublin

Companion Website
www.wiley.com/go/lovric

Protein engineering has proved to be one of the more fruitful technological approaches in biotechnology, being both very powerful and able to generate valuable intellectual property. This book aims to present examples in which the application of protein engineering has successfully solved problems arising in industrial biotechnology. There is a section on its use to enhance purification of recombinant proteins. The use of protein engineering to modify the activity or the stability of industrial enzymes from lipases to proteases, from carboxypeptidases to glucanases and glucosidases, and from pectin modifying enzymes to enzymes able to degrade recalcitrant compounds is extensively covered. It is shown how areas as diverse as agrofood technology, fine chemistry, detergents, bioremediation and biosensors receive significant contributions from protein and solvent engineering. The application of protein engineering to health care is also covered, from the development of new vaccines to new potential therapeutic proteins. A specific notation is given to protein engineering in the development of target molecules for drug discovery. International in scope, the many contributions are drawn from academia and industry. The text should be of interest to students and researchers in industrial biotechnology as well as to everybody interested in basic research in protein structure, molecular genetics, bio-organic chemistry, biochemistry, agrobiotechnology, pharmaceutical sciences and medicine.

Principles of Proteomics, Second Edition, provides a concise and user-friendly introduction to the diverse technologies used for the large-scale analysis of proteins, as well as their applications, and their impact in areas such as drug discovery, agriculture, and the fight against disease. Proteomics is a fast-advancing field in which researchers seek to capture all the proteins in the cell and characterize them in ever more detail. Principles of Proteomics has been fully updated to reflect the most recent developments in the field without losing its focus on the underlying principles. With worked examples, case studies profiling both established and emerging technologies, and further reading lists for each chapter, Principles of Proteomics is an ideal introduction for students, researchers and those working in the industry.

The aim this volume is to present the methods, challenges, software, and applications of this widespread and yet still evolving and maturing field. Computational Protein Design, the first book with this title, guides readers through computational protein design approaches, software and tailored solutions to specific case-study targets. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Computational Protein Design aims to ensure successful results in the further study of this vital field.

This book reviews the current state of epigenetics and proteomics of leukemia and introduces the methods that are important to process and evaluate these factors in leukemia. In particular, epigenetic modifiers and their inhibitors in leukemia treatment as well as approaches to the epigenetic treatment of leukemia are covered. Various computational methods for proteome analysis are also described in detail, including 2DE fractionation and visualization, proteomic data processing, image acquisition and data anlaysis, and more. Protein localization in leukemia is also covered, in addition to the future of leukemia therapy. Epigenetics and Proteomics of Leukemia is an ideal book for advanced biomedical scientists and students, medical doctors and students, bioinformatics and health informatics researchers, computational biologists, structural biologists, systems biologists, and bioengineers.

This volume describes methods and protocols for the fragment-based screening of proteins using Surface Plasmon Resonance (SPR). The initial chapter of the book discusses the principle of SPR for the identification of biomolecular interactions, while the subsequent chapters introduce methods for labelling proteins with different tags including, histidine and biotin tags. It also discusses techniques and factors that affect the amine and biotin-streptavidin coupling and methods to optimize the interactions. Next, it describes fragment preparation for screening in SPR and presents methods to calculate equilibrium dissociation constant (KD) and ligand efficiency (LE). It reviews techniques of next-generation injections that improve the efficiency of the characterization process over traditional SPR by determining the kinetics and affinity in a single step. Finally, the book elucidates a comprehensive yet representative description of challenges associated with the molecular interaction of proteins using SPR.