Advances in fluorescent proteins, live-cell imaging, and superresolution instrumentation have ushered in a new era of investigations in cell biology, medicine, and physiology. From the identification of the green fluorescent protein in the jellyfish Aequorea victoria to the engineering of novel fluorescent proteins, The Fluorescent Protein Revolution explores the history, properties, and applications of these important probes. The book first traces the history of fluorescent proteins and the revolution they enabled in cellular imaging. It then discusses fluorescent proteins with novel photophysical properties. The book also covers several cutting-edge imaging applications. These include superresolution microscopy of cellular fine structures, FRET microscopy to visualize protein interactions and cell-signaling activities inside living cells, photobleaching and photoactivation techniques to visualize protein behaviors, techniques that exploit plant and algal photoreceptors to enable light-regulated control of enzymatic activities, and the noninvasive imaging of tumor-host interactions in living animals. In color throughout, this book presents the fundamental principles and latest advances in the field, including the associated development of imaging techniques that exploit fluorescent proteins. It is accessible to a broad audience, from optical imaging experts to novices needing an introduction to the field.

Protein Methods by Daniel M. Bollag, Michael D. Rozycki, Stuart J. Edelstein

Reviews from the first edition
"...it is very well written...would certainly be of use to undergraduate and postgraduate students entering the complex world of protein preparation and to more experienced scientists entering this field for the first time." —Biotechnology and Applied Biochemistry

"The book presents these topics with loads of practical detail, so that the reader has little need to consult other reference sources to carry out the techniques described. All in all, a useful book..." —Theoretical & Applied Genetics

"...clearly written with protocols that are easy to follow and the text is well spread-out and easy to read...all methods are fully referenced...a useful book for beginners at a reasonable price." —FEBS Letters

This revised and expanded Second Edition of Protein Methods remains the first source for a complete summary of tested and proven protein techniques. Now divided into two parts, the book begins with the essential chapters from the first edition, updated to reflect important changes in methodology. The chapter on protein isolation includes a new section focusing on the isolation of proteins from inclusion bodies. In the second section, four new chapters are devoted to protein purification and crystallization.

Chapters include

• Preparation for Protein Isolation
• Protein Extraction and Solubilization
• Protein Concentration Determination
• Concentrating Protein Solutions
• Gel Electrophoresis Under Denaturing Conditions
• Gel Electrophoresis Under

• reagent items and equipment required for each method
• chapter "view-at-a-glance" tabs
• illustrations for all gel applications and other methods
• complete supplier list
• appendices of basic chemical parameters and molecular data
• key references
• descriptive tables
• Safety notes.

In the mature brain calcium ions play pivotal roles in transmembrane and intracellular transmission of signals. Thus, calcium is involved in numerous neuronal functions including neurotransmitter release, enzyme regulation, modulation of neuronal excitability, gene expression, microtubular transport or synaptic plasticity. Many of these calcium-dependent processes are mediated or modulated by a number of cytosolic calcium-binding proteins. All nerve cells contain the calcium-binding protein calmodulin. Other CaBPs are restricted to certain nerve celltypes, i.e. parvalbumin, calbindin and calretinin.

Recent advances in large-scale DNA sequencing technology have made it possible to sequence the entire genome of an organism. Attention is now turning to the analysis of the product of the genome, the proteome, which is the set of proteins being expressed by a cell. Two-dimensional gel electrophoresis can be used to create cellular protein maps which give a quantitative and qualitative picture of the proteome. Mass spectrometry is the method of choice for the rapid large-scale idenfification of these proteomes and their modifications. An understanding of these methods is critical for scientists in the "Post-Genome" era.

An Introduction to Peptide Chemistry P. D. Bailey, University of York Peptide chemistry is a key area in natural product chemistry, combining aspects of analysis, synthesis and biochemistry. In recent years peptide chemistry has emerged as a discipline in its own right, distinct from amino acid chemistry and protein chemistry. The importance of peptide chemistry is reflected in the intense research interest, exemplified by the progress made in solid-phase peptide synthesis. Recent developments in the determination and prediction of the three-dimensional structure of peptides, and in our understanding and control of their biosynthesis, have led to dramatic advances in the field. This book is intended as a short treatise on peptide chemistry aimed at upper-level undergraduates studying chemistry and biochemistry. This concise account has been thoughtfully presented; emphasis is placed on the principles of peptide chemistry, and how these relate to organic, physical, and biological chemistry.

This book is based on an advanced course of lectures on ribosome structure and protein biosynthesis that I offer at the Moscow State University. These lectures have been part of a general course on molecular biology for almost three decades, and they have undergone considerable evolution as knowledge has been pro gressing in this field. The progress continues, and readers should be prepared that some facts, statements, and ideas included in the book may be incomplete or out of-date. In any case, this is primarily a textbook, but not a comprehensive review. It provides a background of knowledge and current ideas in the field and gives ex amples of observations and their interpretations. I understand that some interpre tations and generalizations may be tentative or disputable, but I hope that this will stimulate thinking and discussing better than if I left white spots. The book has a prototype: it is my monograph "Ribosome Structure and Pro tein Biosynthesis" published by the Benjamin/Cummings Publishing Company, Menlo Park, California, in 1986. Here I have basically kept the former order of pre sentation ofthe topics and the subdivision into chapters. The contents ofthe chap ters, however, have been significantly revised and supplemented. The newly writ ten chapters on translational control in prokaryotes (Chapter 16) and eukaryotes (Chapter 17) are added."

This book will focus on new molecular interactions and novel activities and the associated diseases that have been recently discovered from the studies of eukaryotic and mammalian aminoacyl-tRNA synthetases. In addition, the potential applications of ARS researches in biotechnology and medicine will be addressed.

Molecular farming is a biotechnological approach that includes the genetic adjustment of agricultural products to create proteins and chemicals for profitable and pharmaceutical purposes. Plant molecular farming describes the manufacture of recombinant proteins and other biologically active product in plants. This approach depends on a genetic transformation of plants that can be accomplished by the methods of stable gene transfer, such as gene transfer to nuclei and chloroplasts, and unstable transfer methods like viral vectors. The requirement for recombinant proteins in terms of quality, quantity, and diversity is increasing exponentially This demand is traditionally met by recombinant protein construction technologies and the engineering of orthodox expression systems based on bacteria or mammalian cell cultures. However, majority of developing countries cannot afford the high costs of medicine derived from such existing methods. Hence, we need to produce not only the new drugs but also the cheaper versions of those already present in the market. Plant molecular farming is considered as a cost-effective technology that has grown and advanced tremendously over the past two decades. This book summarizes the advances and challenges of plant molecular farming for all those who are working on or have an interest in this rapidly emerging area of research.

As a scientist with an interest in proteins you will, at some time in your career, isolate an enzyme that turns out to be yellow-or perhaps you already have. Alternatively, you may identify a polypeptide sequence that is related to known flavin-containing proteins. This may, or may not, be your first encounter with flavoproteins. However, even if you are an old hand in the field, you may not have exploited the full range of experimental approaches applicable to the study of flavoproteins. We hope that Flavoprotein Protocols will encourage you to do so. In this volume we have sought to bring together a range of experimental methods of value to researchers with an interest in flavoproteins, whether or not these researchers have experience in this area. A broad range of techniques, from the everyday to the more specialized, is described by scientists who are experts in their fields and who have ext- sive practical experience with flavoproteins. The wide range of approaches, from wet chemistry to dry computation, has, as a consequence, demanded a range of formats. Where appropriate (particularly for analytical methods) the protocol described is laid out in easy-to-follow steps. In other cases (e. g. , the more advanced spectroscopies and computational methods) it is far more apt to describe the general approach and relevance of the methods. We hope this wide-ranging approach will sow the seeds of many future collaborations - tween laboratories and further our knowledge and understanding of how f- voproteins work.

The extracellular matrix is a network of fibres that hold cells together. It is broken down during normal physiological and disease processes such as bone remodelling, embryogenesis, cancer, and arthritis. This book is an up-to-date and comprehensive collection of data about the matrix metalloproteinases - enzymes which breakdown the extracellular matrix, and their specific inhibitors.

This volume contains the proceedings of the Ninth Meeting of the "International Study Group for Tryptophan Research" (lSTRY), held at the University of Hamburg, Germany, from October 10 to 14, 1998. At this meeting the recent developments in the field of tryptophan research were presented by leading researchers from all over the world in 81 oral and 48 poster contri butions. Research on tryptophan and its derivatives provides an inexhaustible subject. At the conference we tried to compose a multifacetted picture of the recent investiga tions through contributions from the major disciplines involved. Thus, we tried to strike a balance between basic research topics and clinical, nutritional or industrial applica tions. We offered workshops on tryptophan (in sleep and mood), melatonjn, IDO-acti vation and the eosinophilia-myalgia syndrome (EMS) as a platform for intensive discussion for the participants. In these proceedings many contributions are multidisciplinary and have practical or theoretical implications for different research fields. Hence, we have organized this volume in nine main chapters according to basic disciplines and subjects. We are aware that this classification is artificial, but we hope that it is the best compromise for contributors and readers."

It is by no means a revelation that proteins are not uniformly distributed throughout the cell. As a result, the idea that protein molecules, because of the specificity with which they can engage in interactions with other proteins, may be aimed-via these interactions-at a restricted target, is a fundamental one in contemporary molecular life sciences. The target may be variously c- ceived as a specific molecule, a group of molecules, a structure, or a more generic type of intracellular environment. Because the concept of protein targeting is intuitive rather than expl- itly defined, it has been variously used by different groups of researchers in cell biology, biochemistry, and molecular biology. For those working in the field of intracellular signaling, an influential introduction to the topic was the seminal article by Hubbard & Cohen (TIBS [1993] 18, 172-177), which was based on the work of Cohen's laboratory on protein phosphatases. Sub- quently, the ideas that they discussed have been further developed and extended by many workers to other key intermediaries in intracellular sign- ing, including protein kinases and a great variety of modulator and adaptor proteins.

Myosins are a diverse superfamily of molecular motor proteins, which share the ability to reversibly bind actin and hydrolyse MgATP. They are capable of either translocating actin filaments or translocating vesicles or other cargo on fixed actin filaments. There are currently 15 distinct classes in the myosins superfamily, based on sequence homology. Myosin II and myosin I proteins are familiar and well studied; while Classes III-XV are less well characterized. All myosins examined to date are multimeric and appear to possess at least three functional domains, a head, neck, and tail. Myosins (second edition) explores the structure and functional properties of myosins, their regulation, and mutational analysis. It has been thoroughly updated since the first edition was published in 1995 including sections on the three additional classes defined by new sequences, information provided by the crystal structure of seven new Dicytostelium motor domains, and data from new techniques such as molecular imaging and tagging proteins with GFP 20. The three human diseases that are now known to be linked to mutations in different myosin heavy or light chains are also covered, including more than 50 mutations associated with hyperotrophic cardiomyopathy.

Biological chemistry is a major frontier of inorganic chemistry. Three special volumes devoted to Metal Sites in Proteins and Models address the questions: How unusual ("entatic") are metal sites in metalloproteins and metalloenzymes compared to those in small coordination complexes? And if they are special, how do polypeptide chains and co-factors control this? The chapters deal with iron, with metal centres acting as Lewis acids, metals in phosphate enzymes, with vanadium, and with the wide variety of transition metal ions which act as redox centres. They illustrate in particular how the combined armoury of genetics and structure determination at the molecular level are providing unprecedented new tools for molecular engineering.

Biological chemistry is a major frontier of inorganic chemistry. Three special volumes devoted to Metal Sites in Proteins and Models address the questions: how unusual ("entatic") are metal sites in metalloproteins and metalloenzymes compared to those in small coordination complexes? And if they are special, how do polypeptide chains and co-factors control this? The chapters deal with iron, with metal centres acting as Lewis acids, metals in phosphate enzymes, with vanadium, and with the wide variety of transition metal ions which act as redox centres. They illustrate in particular how the combined armoury of genetics and structure determination at the molecular level are providing unprecedented new tools for molecular engineering.

Gel Electrophoresis of Proteins: A Practical Approach (third edition) is an up-to-date manual providing details of a variety of methods for running protein gels that will be very widely applicable and an abundance of new techniques and equipment developed since the publication of the second edition. As with the highly successful second edition, this new text places an emphasis on the practical aspects involved in the techniques - presenting them in a clear and accessible format.

The precise shape of a protein is a crucial factor in its function. How do proteins become folded into the right conformation? Molecular chaperones and protein folding catalysts bind to developing polypeptides in the cytoplasm and ensure correct folding and transport. This Guidebook catalogues the latest information on nearly 200 of these molecules, including the important class of heat shock proteins; each entry is written by leading researchers in the field.

Concentrating on the nutrient bioavailability of vitamins, this text provides comprehensive reference material, emphasizing analysis, chemical structure and nomenclature, intestinal absorption and transport, and interaction with other nutrients. The book should be of interest to all those working with vitamins, including biochemists, nutritionists and dieticians, food and feed scientists, medical researchers and in the libraries of all establishments where the subject is studied, researched or taught.

The three-dimensional structure of proteins is a key factor in their biological activity. There is an increasing need to be able to predict the structure of a protein once its amino-acid sequence is known; this book presents practical methods of achieving that ambitious aim, using the latest computer modelling algorithms.

New textbooks at all levels of chemistry appear with great regularity. Some fields like basic biochemistry, organic reaction mechanisms, and chemical thermodynamics are well represented by many excellent texts, and new or revised editions are published sufficiently often to keep up with progress in research. However, some areas of chemistry, especially many of those taught at the graduate level, suffer from a real lack of up-to-date textbooks. The most serious needs occur in fields that are rapidly changing. Textbooks in these subjects usually have to be written by scientists actually involved in the research which is advancing the field. It is not often easy to persuade such individuals to set time aside to help spread the knowledge they have accumulated. Our goal, in this series, is to pinpoint areas of chemistry where recent progress has outpaced what is covered in any available textbooks, and then seek out and persuade experts in these fields to produce relatively concise but instructive introductions to their fields. These should serve the needs of one semester or one quarter graduate courses in chemistry and biochemistry. In some cases the availability of texts in active research areas should help stimulate the creation of new courses.

ism (i. e. , Saccharomyces carlsbergensis, or brewer's yeast) and one of its corresponding enzymes. The experiments on this organism and enzyme are not limited to the materials suggested and can be easily adapted to the desired technical level and available budget. Similarly, the subse- quent cloning experiments suggest that use of particular vectors and strains, but, as indicated, alternative materials can be used to success- fully perform the laboratory exercises. We would like to thank the corporate sponsors of the Biotechnology Training Institute for providing the materials and expertise for the devel- opment of our programs, and thus for the materials in this manual. These sponsors include: * Barnsteadffhermolyne, Dubuque, IA * Beckman Instruments, Somerset, NJ * Bio-Rad Laboratories, Hercules, CA * Boehringer Mannheim Corporation, Indianapolis, IN * Coming Costar Corporation, Cambridge, MA * FMC BioProducts, Rockland, ME * Kodak Laboratory Products, New Haven, CT * Labconco, Kansas City, MO * MJ Research, Cambridge, MA * Olympus Instruments, Lake Success, NY * Pharmacia Biotech, Piscataway, NJ * Savant, Inc. , Farmingdale, NY * VWR Scientific, Philadelphia, P A We would also like to thank the following individuals for their input, comments, and suggestions: Tom Slyker, Bernie Janoson, Steven Piccoli, John Ford,JeffGarelik, Yanan Tian, and Douglas Beecher. Special thanks to Alan Williams for his critique of the chromatography experiments and Shannon Gentile for her work in the laboratory. We would especial- ly like to thank Maryann Burden for her comments and encouragement.

Protein engineering endeavors to design new peptides and proteins or to change the structural and/or functional characteristics of existing ones for specific purposes, opening the way for the development of new drugs. This work develops in a comprehensive way the theoretical formulation for the methods used in computer-assisted modeling and predictions, starting from the basic concepts and proceeding to the more sophisticated methods, such as Monte Carlo and molecular dynamics. An evaluation of the approximations inherent to the simulations will allow the reader to obtain a perspective of the possible deficiencies and difficulties and approach the task with realistic expectations. Examples from the authors laboratories, as well as from the literature provide useful information.

This book collates and reviews recent advances in the microbial metabolism of amino acids, emphasizing diversity - in terms of the range of organisms under investigation and their natural ecology - and the unique features of amino acid metabolism in bacteria, yeasts, fungi, protozoa and nematodes. As well as studying the individual amino acids, including arginine, sulfur amino acids, branched-chain amino acids and aromatic amino acids, a number of themes are explored throughout the work. These include: - Comparative issues between the metabolism of microbes and those of higher organisms, including plants and mammals - Potential for drug targets in pathways of both biosynthesis and degradation of amino acids - Relationship between amino acids or associated enzymes and virulence in parasitic pathogens - Practical implications for food microbiology and pathogen characterization - Future priorities relating to fundamental biochemistry of microrganisms, food quality and safety, human and animal health, plant pathology, drug design and ecology As the volume of research into the metabolism of amino acids grows, this comprehensive study of the subject is a vital tool for researchers in the fields of biological, medical and veterinary sciences, including microbiology, biochemistry, genetics and pathology. This book is also essential for corporate organizations with active research and development programmes, such as those in the pharmaceutical industry.

The intent of this work is to bring together in a single volume the techniques that are most widely used in the study of protein stability and protein folding. Over the last decade our understanding of how p- teins fold and what makes the folded conformation stable has advanced rapidly. The development of recombinant DNA techniques has made possible the production of large quantities of virtually any protein, as well as the production of proteins with altered amino acid sequence. Improvements in instrumentation, and the development and refinement of new techniques for studying these recombinant proteins, has been central to the progress made in this field. To give the reader adequate background information about the s- ject, the first two chapters of this book review two different, yet related, aspects of protein stability. The first chapter presents a review of our current understanding of the forces involved in determining the conf- mational stability of proteins as well as their three-dimensional folds. The second chapter deals with the chemical stability of proteins and the pathways by which their covalent structure can degrade. The remainder of the book is devoted to techniques used in the study of these two major areas of protein stability, as well as several areas of active research. Although some techniques, such as X-ray crystallography and mass spectroscopy, are used in the study of protein stability, they are beyond the scope of this book and will not be covered extensively.

This book reviews the long-standing debate over the relative merits of a high-protein versus a low-protein diet. When protein (or 'animal substance') was first discovered in vegetable foods it was hailed as the only true nutritional principle. Leibig, the leading German chemist of the mid-nineteenth century, believed that it provided the sole source of energy for muscular contraction. In contrast, health reformers argued that high intakes were over-stimulating, leading to dissipation and decline. The subject came to widespread public attention again in the 1950s as the United Nations debated the need for providing protein supplements to Third World infants. At a time when the concern has resurfaced that over-consumption of protein in affluent societies may damage health, this book provides a fascinating historical perspective.