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.

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.

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 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.

For many years, the authors have investigated the adaptive role of heat shock proteins (HSPs) in different animals, including the representatives of homothermic and poikilothermic organisms that inhabit regions with contrasting thermal conditions. This book will summarize the data accumulated in the course of these studies and describe the general molecular mechanisms underlying the adaptation of various organisms to aggressive environments. We also concentrate on different evolutionary trends characteristic for HSP systems in the course of adaptation to fluctuating environmental conditions. In addition, we describe the peculiarities in the regulatory regions of heat shock genes necessary for fine tuning of these systems providing the adaptation to adverse conditions. Special emphasis is given to the role of mobile elements in the evolution and functioning of various groups of HSP genes. The book combines the results of field studies and laboratory analysis of stress genes systems.

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.

Using a novel approach that combines high temporal resolution of the laser T-jump technique with unique sets of fluorescent probes, this study unveils previously unresolved DNA dynamics during search and recognition by an architectural DNA bending protein and two DNA damage recognition proteins. Many cellular processes involve special proteins that bind to specific DNA sites with high affinity. How these proteins recognize their sites while rapidly searching amidst ~3 billion nonspecific sites in genomic DNA remains an outstanding puzzle. Structural studies show that proteins severely deform DNA at specific sites and indicate that DNA deformability is a key factor in site-specific recognition. However, the dynamics of DNA deformations have been difficult to capture, thus obscuring our understanding of recognition mechanisms. The experiments presented in this thesis uncover, for the first time, rapid (~100-500 microseconds) DNA unwinding/bending attributed to nonspecific interrogation, prior to slower (~5-50 milliseconds) DNA kinking/bending/nucleotide-flipping during recognition. These results help illuminate how a searching protein interrogates DNA deformability and eventually "stumbles" upon its target site. Submillisecond interrogation may promote preferential stalling of the rapidly scanning protein at cognate sites, thus enabling site-recognition. Such multi-step search-interrogation-recognition processes through dynamic conformational changes may well be common to the recognition mechanisms for diverse DNA-binding proteins.

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 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.

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.

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."

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.

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.

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.

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 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.

"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."

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."

Harnessing the sun s energy via photosynthesis is at the core of sustainable production of food, fuel, and materials by plants, algae, and cyanobacteria. Photosynthesis depends on photoprotection against intense sunlight, starting with the safe removal of excess excitation energy from the light-harvesting system, which can be quickly and non-destructively assessed via non-photochemical quenching of chlorophyll fluorescence (NPQ). By placing NPQ into the context of whole-organism function, this book aims to contribute towards identification of plant and algal lines with superior stress resistance and productivity. By addressing agreements and open questions concerning photoprotection s molecular mechanisms, this book contributes towards development of artificial photosynthetic systems. A comprehensive picture from single molecules to organisms in ecosystems, and from leading expert s views to practical information for non-specialists on NPQ measurement and terminology is presented."

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.

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.

Adhesion plays a major role in the bacterial lifestyle. Bacteria can adhere to organic and inorganic surfaces, to each other, and of course to host cells during pathogenesis. The focus of this book is: how are such adhesion phenomena best studied? Microbial genetics experiments have greatly enhanced our knowledge of what bacterial factors are involved in adhesion. For numerous reasons, though, biochemical and structural biology knowledge of the molecular interactions involved in adhesion are limited. One major problem has been a lack of interdisciplinary research and understanding in the field. On the one hand, the microbiologists lack detailed knowledge of the biophysical possibilities and have limited access to the frequently expensive instrumentation involved while on the other hand, the experts in these methods frequently do not have access to the biological materials, nor do they necessarily understand the biological questions to be answered. The purpose of this book is thus to overcome this gap in communication between researchers in biology, chemistry and physics and to display the many ways and means to investigate bacterial adhesion. We hope to stimulate new and ground-breaking research.

Proteases form one of the largest and most diverse families of enzymes known. Once considered primarily as "enzymes of digestion," it is now clear that proteases are involved in every aspect of cellular function. Members of the diverse families of proteases act to promote cellular proteolysis found in nature, and their deregulation may result in different pathophysiological conditions, such as tumor progression, vascular remodeling, atherosclerotic plaque progression, ulcer, rheumatoid arthritis, and Alzheimer's disease. Many micro-organisms require proteases for replication or use proteases as virulence factors, which have facilitated the development of protease-targeted therapies for a variety of parasitic diseases. Proteases in Health and Disease represents a comprehensive overview of the fascinating field of proteases by various renowned experts, and focuses on the recently elucidated functions of complex proteolytic systems in physiology and pathophysiology. Part A, Molecular and Biochemical Aspects of Proteases, illustrates some of the major proteases, such as calpains, matrix metalloproteases, fibrinolytic serine proteases, and aspartic proteases, which play a significant role in a variety of pathologies and may be a target for therapy either by their up regulation or down regulation. Part B, Involvement of Proteases in Diseases Processes, deals with the functional roles of the individual proteases in the progression of diseases such as cardiovascular and inflammatory lung disease, malaria, cholera, autism spectrum disorder, hepatitis, and ischemia-reperfusion injury induced cardiac diseases. With this multi-disciplinary scope, the book bridges the gap between fundamental research and biomedical and pharmaceutical applications, making this a thought-provoking reading for basic and applied scientists engaged in biomedical research.