This book provides a molecular view of membrane transport by means of numerous biochemical and biophysical techniques. The rapidly growing numbers of atomic structures of transporters in different conformations and the constant progress in bioinformatics have recently added deeper insights.The unifying mechanism of energized solute transport across membranes is assumed to consist of the conformational cycling of a carrier protein to provide access to substrate binding sites from either side of a cellular membrane. Due to the central role of active membrane transport there is considerable interest in deciphering the principles of one of the most fundamental processes in nature: the alternating access mechanism.This book brings together particularly significant structure-function studies on a variety of carrier systems from different transporter families: Glutamate symporters, LeuT-like fold transporters, MFS transporters and SMR (RND) exporters, as well as ABC-type importers.The selected examples impressively demonstrate how the combination of functional analysis, crystallography, investigation of dynamics and computational studies has made it possible to create a conclusive picture or more precisely, a molecular movie . Although we are still far from a complete molecular description of the alternating access mechanism, remarkable progress has been made from static snapshots towards membrane transport dynamics."

The adsorption of proteins at interfaces plays a role in many ?elds, such as health, food, environment and analysis. Fundamental aspects are useful when considering applications. We focus here especially on solid-liquid interfaces and present a few fundamental studies regarding adsorption - netics and conformational changes, and examples of applications to sensors and membranes. The ?rst part is dedicated to fundamental studies performed using - tical waveguide lightmode spectroscopy, as an example of a technique that has the advantage of not requiring labelled proteins, but is limited to s- ci?c supports. Conversely, the radiolabelling of proteins, which has the disadvantage of any labelling process, allows application to any kind of s- faces. As proteins bear both positive and negative charges, we can expect thein?uenceofanelectric?eldnormaltothe interfaceonthe pack- ing order at interfaces. The re?ning of data treatment may also lead to the determination of useful structural parameters. The balance between protein-surface and protein-protein interactions is a key point for the - scription of the structure at high coverage of the surface. Electrokinetic methods, like measurement of the streaming potential, may be helpful in the electrical characterisation of the interfacial layer facing the solution. The second part includes different bench techniques that were dev- oped to improve the sensitivity of the characterisation of the orientation and structure of the proteins at interfaces: dual polarisation interferometry and total internal re?ection ellipsometry are such recent examples.

This volume explores the considerable efforts that have been directed towards the development of G Protein-Coupled Receptors (GPCR) screening assays in order to disclose GPCR acting compounds, elucidate signaling mechanisms or evaluate compound's efficacy. New discoveries in the field, along with the widely recognized need for better and safer pharmaceutical drugs constitute the main motivation for this book. Readers, both beginners and experienced researchers, will receive an updated overview of not only the established, but also the innovative technologies that promise to advance GPCR drug research. This book is organized into two major parts: the introductory part discusses the necessary foundations for the understanding of GPCR action and the rationale behind the design of the available screening assays; and part two provides detailed protocols for different screening approaches. Written in the highly successful Methods in Molecular Biology series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting optimal results in the laboratory. Practical and innovative, G Protein-Coupled Receptor Screening Assays: Methods and Protocols reaches out to everyone involved in the discovery of GPCR-active drugs, and provides a transversal overview of the different levels of GPCR signaling addressable in the different screening strategies and presents practical examples of how current assay technologies are contributing to new paradigms in GPCR drug research.

This volume provides a comprehensive list of protocols for molecular biologists, biochemists and geneticists. Chapters cover protocols that further the study into protein complexes that modify chromatin either by adding or removing post-translational modifications, or by exchanging histone variants within the nucleosome. Written in the highly successful Methods in Molecular Biology 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 cutting-edge, Histones: Methods and Protocols aims to ensure successful results in the further study of this vital field.

This book addresses the most recent advances in the transport of proteins across a variety of biological membranes. In addressing this topic, this volume includes several new twists not previously addressed in the literature. In the last few years, the study of protein translocation has been revolutionized by the availability of structural information on many of the components and complexes involved in the process. Unlike earlier books written on protein translocation, this volume considers these advances. In addition, several chapters discuss facets of protein translocation from a systems biology perspective, considered by many to be the next paradigm for biological study. Readers of this book will come away with a deeper understanding of the problems facing researchers of protein translocation and see how the most modern biological techniques and approaches are being recruited to answer those questions. The chapters are also written such that problems awaiting future investigation are clearly presented.

Prions are infectious, self-propagating proteinaceous agents that cause fatal neurodegenerative diseases, including Creutzfeldt-Jakob Disease (CJD) in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE) in cattle. In recent years great strides have been made in our understanding of the mechanism of prion propagations and neurotoxicity, however much remains to be discovered. In this book, renowned prion experts review the most recent advances to provide an overview of the field.

This volume provides the reader with detailed protocols that describe a variety of in vitro and in vivo techniques that study reductionist systems and human samples. The methods covered in this book explore a broad selection of topics such as functional aspects of alpha-synuclein biology, its lipid-interactions and misfolding, and functional deficits in cells and mice. Chapters also cover topics such as kinetic measurements of endocytosis and exocytosis in cultured neurons; electrochemiluminescence-based detection; yeast-based screens to target alpha-synuclein toxicity; mass-spectrometry of alpha-synuclein in rat cortical neurons; and expression and purification of untagged a-synuclein. Written in the highly successful Methods in Molecular Biology 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. Cutting-edge and thorough, Alpha-Synuclein: Methods and Protocols is a valuable reference guide that aids researchers working on topics related to alpha-synuclein and its various aspects.

In the late 1980s, Peptide Societies were established in Europe, the United States, and Japan, and more recently, in the Asian and the Pacific Rim regions including Australia, China, and Korea. At the time of the establishment of the American, European and Japanese Peptide Societies, the International Liaison Organizing Committee representing these Peptide Societies, along with the Australian Peptide Society, began discussions for holding international confer ences which would supercede or be held in lieu of the numerous individual meetings, held by the peptide societies of each individual country or region. The representative of the Chinese Peptide Society participated in these discus sion in the International Liaison Organizing Committee at the meeting of the American Peptide Symposium in Nashville, in June 1997. After lengthy discus sions over several years, we agreed to organize and host the International Peptide Symposium in Japan. The First International Peptide Symposium (IPS'97) was held on November 30-December 5, 1997, in Kyoto, and was co sponsored by four Peptide Societies. The attendance at this Symposium was 550 participants, including representatives from 32 different countries. We were very pleased with this outcome and anticipate an even larger attendance for forthcoming Symposia in future years. The revolution and advances in science and technology during the past two decades has caused traditional peptide chemistry to expand to peptide science, spreading from physical science to biology, pharmacology, and medicine.

This book provides an up-to-date and comprehensive overview of protein phosphatase research, a rapidly evolving field with increasing importance in our understanding of the molecular basis of cell biology and of pathological processes.

The book covers dephosphorylation processes involving serine/threonine, as well as tyrosine and histidine residues, and aims to be a useful resource for both the advanced reader as well as the newcomer to the field. It is also valuable for those working in the pharmaceutical and Biotech industries.

It is now widely accepted that the extracellular matrix (ECM) is a key determinant of tissue-specific gene expression. Signals provided by ECM are transduced by integrins, a large and growing superfamily of transmembrane heterodimeric cell surface receptors that link the ECM to structural and fu- tional elements within the cell. A wide range of cellular phenotypes have been shown to be regulated by integrins, including growth, differentiation, mig- tion, invasion, angiogenesis, and apoptosis. Furthermore, abnormalities of integrin expression and function have been implicated in the etiology of va- ous pathologic conditions, including cardiovascular disease, inflammatory disorders, and cancer. Thus integrins have emerged as an important class of molecules with wide ranging implications for understanding basic biological processes. In Integrin Protocols we provide a wide-ranging collection of laboratory protocols intended to assist investigators interested in integrins in working productively with these molecules, in studying their expression, and in pot- tially manipulating that expression to define their role(s) in relevant biolo- cal models. Protocols are provided for the analysis of integrin expression both at the RNA and protein levels (Chaps. 2, 5, and 7). Delcommenne and Streuli describe procedures for making rat monoclonal antibodies specific for mouse integrins; Schneller et al. and Arap and Huang describe methods for western blotting of integrins and RT-PCR analysis. Protocols are included that cover the analysis of the functional properties of integrins (Chaps. 1, 3, 4, 8, and 9 through 11). Koivunen et al.

This book covers elements of both the data-driven comparative modeling approach to structure prediction and also recent attempts to simulate folding using explicit or simplified models. Despite the unsolved mystery of how a protein folds, advances are being made in predicting the interactions of proteins with other molecules. Also rapidly advancing are the methods for solving the inverse folding problem, the problem of finding a sequence to fit a structure. This book focuses on the various computational methods for prediction, their successes and their limitations, from the perspective of their most well known practitioners.

This volume provides a comprehensive guide on the Cyr61 (Cysteine-rich 61) (CCN) family of proteins and genes from basic research to cutting-edge methodologies and state-of-the-art techniques. Chapters details practical tips to overcome any obstacles with experimentation pertaining to chemistry, biology, physiology, pathology, medical and dental sciences and pharmacology of CCN proteins. Written in the highly successful Methods in Molecular Biology 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 cutting-edge, CCN Proteins: Methods and Protocols will be a valuable resource for a wide audience, ranging from the experienced CCN researchers looking for new approaches to junior graduate students just beginning in CCN research.

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.

"Protein Affinity Tags: Methods and Protocols" provides researchers with the necessary information, tools, and strategy required for proper inquiry into a given protein s function and structure. Today s researchers can easily alter the amino acid sequence of any given protein, a powerful technology allowing for the precise engineering of specific proteins. Protein affinity tagging employs the use of known protein binding interactions in order to fish out a protein-of-interest or, with more advanced tags, a protein complex. This technology paired with recent advancements in DNA sequencing technology promises a future that is awash with novel genomic information information that will not only expand our knowledge of the processes that govern life, but also fuel innovation that will undoubtedly conquer many of today s most salient health and environmental challenges. Written in the successful "Methods in Molecular Biology" 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, "Protein Affinity Tags: Methods and Protocols" will be an invaluable resource to those seeking to employ protein affinity tags to study their biological system of interest."

Milestones in the techniques and methodology of polypeptide structure determination include the determination of the sequence of insulin by Sanger in 1951 (I) and the introduction of the repeti tive degradation of proteins with phenylisothiocyanate by Edman in 1959 (2). The automation of Edman chemistry (3) played a major role in the determination of polypeptide structures. Important modifications of Edman chemistry include the solid-phase approach by Laursen in 1971 (4) and the use of modified Edman reagents such as 4-N, N-dimethylaminoazobenzene-4'-isothiocy- ate (DABITC) for manual sequencing by Chang et al. (5) in 1976. A second major breakthrough in the analysis of polypeptides was automated amino acid analysis described by Spackman et al. in 1958 (6). However, during the period from 1975 to 1980, it became increasingly clear that the amount of material required for struc tural analysis was more than could be easily isolated for the vast majority of proteins. The field was criticized for its lack of sensitive techniques for the analysis of growth factors, immune modulators, membrane receptors, and peptide hormones. In addition, very little had been done to modernize and improve the original instruments introduced in the mid-1960s. The first indications of improved instrumentation for Edman chemistry came from Wittmann-Liebold's laboratory (7), followed by the introduction of a "micro" sequencer by Hunkapiller and Hood in 1978 (8). The movement toward improved instrumentation culminated in the "gas"-phase sequencer of Hewick et al. (9) in 1981."

Featuring a diverse array of model organisms and scientific techniques, Sirtuins: Methods and Protocols collects detailed contributions from experts in the field addressing this vital family of genes. Opening with methods to generate sirtuin biology tools, the book continues by covering methods to identify sirtuin substrates, to measure sirtuin activity, and to study sirtuin biology. Written in the highly successful Methods in Molecular Biology 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. Comprehensive and easy to use, Sirtuins: Methods and Protocols presents detailed protocols for sirtuin research that can be followed directly or modified to investigate new areas of sirtuin biology.

Since its ?rst description in 1942 in both serum and cerebrospinal ?uid, transthyretin (TTR) has had an eventful history, including changes in name from "prealbumin" to "thyroxine-binding prealbumin" to "transthyretin" as knowledge increased about its functions. TTR is synthesised in a wide range of tissues in humans and other eutherian mammals: the liver, choroid plexus (blood- cerebrospinal ?uid barrier), retinal pigment epithelium of the eye, pancreas, intestine and meninges. However, its sites of synthesis are more restricted in other vertebrates. This implies that the number of tissues synthesising TTR during vertebrate evolution has increased, and raises questions about the selection pressures governing TTR synthesis. TTR is most widely known as a distributor of thyroid hormones. In addition, TTR binds retinol-binding protein, which binds retinol. In this way, TTR is also involved with retinoid distribution. More recently, TTR has been demonstrated to bind a wide variety of endocrine disruptors including drugs, pollutants, industrial compounds, heavy metals, and some naturally occurring plant ?avonoids. These not only interfere with thyroid hormone delivery in the body, but also transport such endocrine disruptors into the brain, where they have the potential to accumulate.

In this volume, expert practitioners present a compilation of methods of functional data analysis (often referred to as "systems biology") and its applications in drug discovery, medicine, and basic disease research. It covers such important issues as the elucidation of protein, compound and gene interactions, as well as analytical tools, including networks, interactome and ontologies, and clinical applications of functional analysis. As a volume in the highly successful Methods in Molecular Biology series, this work provides detailed description and hands-on implementation advice. Reputable, comprehensive, and cutting-edge, Biological Networks and Pathway Analysis presents both "wet lab" experimental methods and computational tools in order to cover a broad spectrum of issues in this fascinating new field.

This book is dedicated to the characterization of peptides and their applications for the study of biochemical systems. The contributing authors are all leaders in the field of peptide research. Part I, Characterization, presents the most recent advances in select analytical techniques. Part II, Application, presents a variety of specific applications for synthetic peptides. This book is an indispensable aid in the pursuit of new directions in peptide research.

A major mechanism by which cells regulate protein function is to place phosphate groups on serine and threonine residues. Though the steady-state level of protein phosphorylation depends on the relative activities of both kinases and phosphatases, a much greater effort has previously gone into the study of the former that the latter . Today, however, there is an increasing appreciation for the role that protein phosphatases play in the dynamic p- cess of protein phosphorylation . To date, there are four major types of protein serine/threonine phosphatase catalytic subunits, designated protein phosphatase type 1, 2A, 2B, and 2C . Each has been identified by the techniques of protein chemistry and enzymology and can be distinguished from one another by their preference for specific substrates as well as their sensitivity to certain acti- tors and inhibitors . Protein Phosphatase Protocols has been assembled in response to the growing interest these enzymes are receiving . The goal of this compilation is to provide a "how-to" experimental guide to aid newcomers as well as s- soned veterans in their research endeavors, thus further contributing towards our ever increasing knowledge of serine/threonine phosphatases . What you have before you contains contributions by many of the current and emerging leaders in the field . To highlight just a few, these chapters c- tain step-by-step information on how to isolate novel phosphatases and re- latory subunits, assay for activity, and generate immunological reagents for both biochemical and biological characterization of these enzymes .

Considerable effort and time is allocated to introducing cell culture and fermentation technology to undergraduate students in academia, generally through a range of courses in industrial biotechnology and related disciplines. Similarly, a large number of textbooks are available to describe the appli- tions of these technologies in industry. However, there has been a general lack of appreciation of the significant developments in downstream processing and isolation technology, the need for which is largely driven by the stringent re- latory requirements for purity and quality of injectable biopharmaceuticals. This is particularly reflected by the general absence of coverage of this s- ject in many biotechnology and related courses in educational institutions. For a considerable while I have felt that there is increasing need for an introductory text to various aspects of downstream processing, particularly with respect to the needs of the biopharmaceutical and biotechnology ind- try. Although there are numerous texts that cover various aspects of protein purification techniques in isolation, there is a need for a work that covers the broad range of isolation technology in an industrial setting. It is anticipated that Downstream Processing of Proteins: Methods and Protocols will play a small part in filling this gap and thus prove a useful contribution to the field. It is also designed to encourage educational strategists to broaden the coverage of these topics in industrial biotechnology courses by including accounts of this important and rapidly developing element of the industrial process.

Membrane proteins play a key role in numerous pathologies such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, and Alzheimer s disease, yet studies on these and other disorders are hampered by a lack of information about the proteins involved. In Heterologous Expression of Membrane Proteins: Methods and Protocols, expert researchers provide an overview of the different heterologous expression systems available to produce these proteins for structural analysis, helping us to understand how these proteins function, and additionally, how their functions can be modified through drug treatment. Chapters examine membrane protein solubilization, purification, and instability in solution, and investigate the strategies that helped to determine the structure of the first heterologously expressed mammalian membrane proteins. Composed in the highly successful Methods in Molecular Biology series format, each chapter contains a brief introduction, step-by-step methods, a list of necessary materials, and a Notes section which shares tips on troubleshooting and avoiding known pitfalls.

Far-reaching and innovative, Heterologous Expression of Membrane Proteins: Methods and Protocols, is an essential guide for novice and experienced researchers alike, providing detailed methods for existing expression systems alongside cutting-edge strategies for future use in the field."

This detailed volume assembles comprehensive protocols to assist with the study of structural, molecular, cell biological, and in vivo facets of GPCRs, and to enable the development of experimental tools for screening novel GPCR drugs. Sections explore the tweaking of ligands, bioluminescence and FRET approaches, specific GPCR signaling properties, as well as visualization of subcellular compartmentalization. Written for the highly successful Methods in Molecular Biology series, 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 practical, G Protein-Coupled Receptor Signaling: Methods and Protocols serves as an ideal reference for life scientists working in a variety of research fields including molecular pharmacology, cell and developmental biology, brain behavior and physiology, drug development and screening. Chapter 4 is available open access under a CC BY 4.0 license via link.springer.com.

This interdisciplinary volume collates research work on kinesins and cancer. Authors attempt to validate members of the kinesin superfamily as potential targets for drug development in cancer chemotherapy. The work begins by highlighting the importance of kinesins, summarising current knowledge and how they are shown to be crucial for mitosis. Chapters go on to explore how this family of proteins are emerging as a novel target for chemotherapeutic intervention and drug development. Readers will learn how kinesins travel along microtubules to fulfill their many roles in intracellular transport or cell division. Several compounds that inhibit two mitotic kinesins (called Eg5 and CENP-E) have entered Phase I and II clinical trials and are explored in these chapters. Additional mitotic kinesins are currently being validated as drug targets, raising the possibility that the repertoire of kinesin-based drug targets may expand in the future. The book is suitable as a reference standard for the field of kinesins and cancer. It will interest those in academia and pharmaceutical companies, and anyone with an interest in the medical relevance of these proteins, which cutting edge methodologies are now enabling us to understand in astonishing detail.

Glutamine is the most abundant amino acid and is a major contributor to whole body nitrogen metabolism and is considered to be "conditionally essential." Glutamine in Health and Disease presents the application of current nutritional knowledge by physicians and dietitians and incorporates emerging fields of science and important discoveries. Section 1 covers glutamine structure and function, glutamine synthetase, glutamine binding protein, glutamine transport, glutamine-rich activation domains and transcription, glutamine transaminase and cell biochemistry. Section 2 covers glucose-independent glutamine metabolism, intestinal barrier function, thyroid-stimulating hormone, glutamine resonances, focal ischemia, plasma glutamine, metabolic stress, cancer and absorption. Section 3 covers dipeptide-bound glutamine, DNA protection, oxidative stress, NF-KB, the inflammatory response, the lung, kidney, GI tract and liver, autophagy, ethanol and diabetes. Finally, Section 4 covers the use of glutamine in preoperative states, enteral and parenteral nutrition, pulmonary infections, cancer, hypoxic injury, arginyl-glutamine, paediatrics, pancreatic surgery, the elderly, gastric emptying gastric bypass and use glutamine cocktails. Written by authors of international and national standing, leaders in the field and trendsetters, Glutamine in Health and Disease is essential reading for nutritionists and dietitians, public health scientists, physicians, epidemiologists, policy makers, and health care professionals of various disciplines.