In this present volume, different approaches are detailed to produce membrane proteins, purify them, study their function, determine their structure, and model them in membrane. Since every membrane protein behaves mostly in a unique way /fashion, knowledge of guidelines and tricks may help to increase chances to express, purify and characterize a peculiar membrane protein. Production of correctly folded protein remains a challenge. Moreover, getting a functional and stable protein requires to optimize membrane mimicking environments that can be detergent or artificial membranes. In some cases, the finding of the correct ligand which will stabilize the desired conformation is needed. In other cases, stabilization can be obtained using specific antibodies. This volume also presents different techniques to analyze the functional status of membrane proteins. Written in the highly successful Methods in Molecular Biology series format, chapters in Membrane Protein Structure and Function Characterization: Methods and Protocols provide different techniques to analyze the functional and structural status of membrane proteins. 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, Membrane Protein Structure and Function Characterization: Methods and Protocols aims to ensure successful results in the further study of this vital field.

This book explores the remarkable information correspondences and probability structures of proteins. Correspondences are pervasive in biochemistry and bioinformatics: proteins share homologies, folding patterns, and mechanisms. Probability structures are just as paramount: folded state graphics reflect Angstrom-scale maps of electron density. The author explores protein sequences (primary structures), both individually and in sets (systems) with the help of probability and information tools. This perspective will enhance the reader's knowledge of how an important class of molecules is designed and put to task in natural systems, and how we can approach class members in hands-on ways.

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

Protein tyrosine phosphatases remove phosphates from the phosphotyrosine residues of target proteins and reverse the action of various protein tyrosine kinases. This essential interplay between the opposing actions of protein tyrosine phosphatases and protein tyrosine kinases forms the basis of signaling networks that underlie the cellular workings of human physiology. Initially passed-off as housekeeping genes; these proteins were only acknowledged to maintain a steady background of phosphotyrosine levels in the cell. However, recent progress in studying their role in embryonic development and human disease has established their importance as regulators of signal regulation. Convincing evidence shows the role of mutations in these proteins to cause and/or intensify the severity of various diseases including metabolic and neurological disorders and also cancer. Protein tyrosine phosphatases have slowly, yet convincingly become crucial targets for therapeutic intervention of various human pathophysiologies. This book describes these signaling enzymes using the molecular details of their structure and mechanistic function. Various subtypes of cysteine-based Class I, II, III and the Haloacid dehalogenase related Class IV protein tyrosine phosphatases have been illustrated and explained. The superfamily of proteins is also described vis-a-vis its complimentary protein phosphoserine/phosphoserine phosphatases. Membrane bound receptor forms and the cytosolic non-receptor protein tyrosine phosphatases have been described for their biological function. This book serves as a reference for any reader looking to understand the sequence features, structural elements, molecular mechanism and cellular function of this superfamily of signaling enzymes.

Recent research suggests that adult growth hormone (GH) deficiency, whether of pathological or physiological origin, is associated with a dis tinct syndrome that includes alterations in body composition, endocrine metabolic function, immune competence, and physical and psychosocial well-being. Not surprisingly, substantial investigative effort is currently focused on validating the above hypothesis and on determining whether restoration of a normal GH-IGF-I axis in various states of adult GH deficiency is clinically useful, safe, and cost-beneficial. This book contains the proceedings from the Symposium on GHRH, GH, and IGF-I: Basic and Clinical Advances, held December 9 to 12, 1993, in San Diego, California, and sponsored by Serono Symposia USA, Inc. The conference was meant to highlight selected novel and exciting clinical research developments related to possible therapeutic uses of recombinant human GH and IGF-I, GHRH, GH releasing peptides, and related GH secretagogues. This meeting occurred only one year after a similar Serono symposium that was somewhat more oriented to novel basic science discoveries, thus attesting to the current scientific and clinical interest in this area."

This new edition provides updated and novel protocols of neuroproteomics methods that encompass both global-scale as well as targeted and specialized topics, which are timely additions for the molecular and phenotypic analysis of the central nervous system and CNS-related disorders. The detailed contents of this book include the exploration of several exciting areas of advanced methods used for neuroproteomics research including relative and absolute protein quantitation by mass spectrometry, characterization of post-translational modifications, as well as bioinformatics and computational approaches. Written for the highly successful Methods in Molecular Biology series, methodology chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and tips on troubleshooting and avoiding known pitfalls. Updated and accessible, Neuroproteomics: Methods and Protocols, Second Edition serves researchers and clinical scientists involved in the area of biomarker research and protein biochemistry, as well as molecular biologists and biochemists who have been involved in proteomics research already or even for those new to the field.

Fourier Transform Infrared microspectroscopy (FTIR) was first developed by William Coblentz in 1905 for analytical purposes. It has been established as a powerful analytical method to analyze a wide range of materials. The most convenient way to analyze the molecular structure was to prepare KBr pellets with small amount of chemical species. Currently, the development of the Universal Attenuated Total Reflectance (UATR) allows the use of ZnSe-Diamond crystal to acquire FTIR spectra directly from the sample with no special preparation. These traditional FTIR analyses have been made with devices capable of performing single measurements, thus, providing a single IR spectrum of the sample. Recent major technological development in FTIR instrumentation was development of microscopes and imaging systems. These devices are now capable of imaging larger sample area, providing not only spectroscopic information but also spatial distributional information. In addition, the development of Focal Point Array (FPA) has made FTIR imaging an emerging area of chemical imaging research. The aim of this book is to summarize in a single document the research work that is being performed using UATR and IR imaging in selected emerging applications in plant materials and biological samples. This book provides the readers new knowledge, updates information, emerging applications, and understanding of the potential use of FTIR Microspectroscopy.

The book addresses the most recent developments in structural and functional proteomics underlying the recent contributions given in these areas by our laboratory to the instrumentations, the methods and the procedures as mutuated from the nanoscale sciences and technologies. These developments introduced in the last few years make now possible protein massive identification (mass spectrometry and biomolecular arrays down to nanoamounts) and protein structural characterization in solution and in crystals down to the atomic scale to an extent and to a degree so far unmatched. Emphasis is placed in the growth by nanobiofilm template of protein crystals of any type and size from millimeter to micron, leading in combination with microfocus synchrotron technology and atomic force microscopy to the definition of a new field called nanocrystallography. The few useful examples being shown, concerning yet structurally unsolved proteins, point this very promising approach nanotechnology-based in structural proteomics using highly focused X-rays. This has not to be confused with the important study of nanocrystals, both organic and inorganic, and novel diamond like nanocomposite materials and devices having 3D protein crystals as matrices to be equilibrated with nanoparticles/gold/silver to be utilized in the most diversified electronic applications here also summarized. vii Acknowledgments We are particularly grateful to Giuseppe Zanotti at the University of Padova for his fundamental collaboration during all the crystallographic studies.

This book covers the new Omics area, Metallomics. As Metallomics is intrinsically a transdisciplinary area, this book is authored by experts in the field on such diverse topics as Environmental, Nuclear, and Human Metallomics. Within these topics metals play important role, as being part of biomolecules, controlling different biochemical process, being signaling agents, being catalyst of biochemical reactions, among others. This volume demonstrates the importance of more investigation about metals and their interactions with biomolecules. As the knowledge in this field is growing and growing daily, then new challenges concerning studies involving Metallomics is appearing, such as comparative metallomics, speciation metallomics, real-time metallomics, new predictions of metals in biomolecules, metalloprotein databank expansion, interactions between metalloprotein-metalloprotein, among others.

Methods of proteomics have been shown to be powerful tools in search of target proteins - proteins that respond in cells to an internal or an external stimulus. Proteomics is widely used in biomedical research. However, in radiation biology research, following exposures of living matter to low doses of either ionizing or non-ionizing radiation, proteomics approach is only very slowly gaining support. This book, by presenting the current status of the use of proteomics in radiation biology, will help to attract attention to the field of radiation proteomics.

This second edition focuses on various techniques to investigate aspects of the TNF Superfamily members in health and disease. Chapters detail protocols on the signaling process of TNF family members, technical examples to investigating the role of TNF family members in physiopathologies, modulation of TNF signaling by pathogens, experimental applications of TNF-reporter mice, methodologies for various assays of TNF family members and the production of recombinant molecules. 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 accessible, The TNF Superfamily: Methods and Applications, Second Edition serves to aid researchers investigating this key family of proteins.

Techniques in the neurosciences are evolving rapidly. There are currently very few volumes dedicated to the methodology - ployed by neuroscrentists, and those that are available often seem either out of date or limited in scope. This series is about the methods most widely used by modern-day neuroscientists and is written by their colleagues who are practicing experts. Volume 1 will be useful to all neuroscientists since rt concerns those procedures used routmely across the wrdest range of s- disciplines. Collecting these general techniques together in a single volume strikes us not only as a service, but will no doubt prove of exceptronal utrlitarran value as well. Volumes 2 and 3 describe current procedures for the analyses of amines and their metabolites and of amino acids, respectively. These collections will clearly be of value to all neuroscrentists working m or contemplating research m these fields. Similar reasons exist for Volume 4 on receptor binding techniques, since experimental details are provided for many types of hgand-receptor binding, including chapters on general prin- ples, drug discovery and development, and a most useful app- dix on computer programs for Scatchard, nonlinear, and compe- tive displacement analyses. Volume 5 provides procedures for the assessment of enzymes involved in neurotransmitter synthesis and catabolism. Volumes in the NEUROMETHODS series will be useful to neurochemists, -pharmacologists, -physiologrsts, -anatomrsts, psychopharmacologists, psychiatrists, neurologists, and chemists (organic, analytical, pharmaceutical, medicinal); in fact, everyone involved m the neuroscrences, both basic and clmical.

Phospholipases generate lipid signaling molecules through their hydrolytic action on phospholipids and are known to regulate function of a variety of cells under normal and diseased conditions. While several physiological, biochemical and molecular techniques have identified key players involved in different disease processes, phospholipases have also emerged as critical players in the pathogenesis of a number of different diseases including cancer and heart disease. In addition, phospholipases are also implicated in such conditions as brain disorder/injury, kidney and immune cell dysfunction. Phospholipases in Health and Disease is a compilation of review articles dedicated to the study of the field with respect to biochemical and molecular mechanisms of normal and abnormal cell function. The wide range of area covered here is of interest to basic research scientists, clinicians and graduate students, who are engaged in studying pathophysiological basis of a variety of diseases. Furthermore, this book highlights the potential of the different phospholipases as therapeutic targets as well as part of prevention strategies. Twenty three articles in this book are organized in four sections that are designed to emphasize the most characterized forms of the phospholipases in mammalian cells. The first section discusses general aspect of phospholipases. Section two covers the role and function of phospholipase A in different pathophysiological conditions. The third section is focussed on phospholipase C which is believed to play a central role in transmembrane signaling. The final section covers phospholipase D which is present in a variety of different cells. The book illustrates that the activation of phospholipases is of fundamental importance in signal transduction affecting cell function. Overall, this book discusses the diverse mechanisms of phospholipase mediated signal transduction in different pathophysiological conditions and raises the possibility of specific forms of phospholipases serving as novel targets for drug development.

Now in its third edition and supplemented with more online material, this book aims to make the "new" information-based (rather than gene-based) bioinformatics intelligible both to the "bio" people and the "info" people. Books on bioinformatics have traditionally served gene-hunters, and biologists who wish to construct family trees showing tidy lines of descent. While dealing extensively with the exciting topics of gene discovery and database-searching, such books have hardly considered genomes as information channels through which multiple forms and levels of information have passed through the generations. This "new bioinformatics" contrasts with the "old" gene-based bioinformatics that so preoccupies previous texts. Forms of information that we are familiar with (mental, textual) are related to forms with which we are less familiar (hereditary). The book extends a line of evolutionary thought that leads from the nineteenth century (Darwin, Butler, Romanes, Bateson), through the twentieth (Goldschmidt, White), and into the twenty first (the final works of the late Stephen Jay Gould). Long an area of controversy, diverging views may now be reconciled.

This book updates the latest development in production, stabilization and structural analysis techniques of membrane proteins. This field has made significant advances since the elucidation of the first 3-D structure of a recombinant G Protein Coupled Receptor (GPCR), rhodopsin, with the structure of several more GPCRs having been solved in the past five years. In fact, the 2012 Nobel Prize in Chemistry was awarded for groundbreaking discoveries on the inner workings of GPCRs. This book is essential reading for all researchers, biochemists and crystallographers working with membrane proteins, who are interested by the structural characterization of their favorite protein and who wish to follow the expression, migration, modifications and recycling of a membrane protein.

Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins provides a detailed guide to the principles and practice of using transgenic plants as a system for the production of heterologous proteins. It is unique in that it covers the complete process of heterologous protein production in plants, from the initial transformation of the plant, through to transcription, transgene stability and finally the downstreaming processing events for protein purification. Written by an international team of industrialists and academics, this book describes:

• the fundamental issues associated with expressing heterologous proteins in plants;
• a number of detailed examples of the successful small-and large-scale production of proteins;
• the essentials of patenting; and
• the commercial exploitation.

This book provides a state-of-the-art report on our current understanding of aquaporins and the future direction of the field. Aquaporins (AQPs) are a group of water-channel proteins that are specifically permeable to water and other small molecules, such as glycerol and urea. To date thirteen water-channel proteins (AQP0 - AQP12) have been cloned and the mechanisms and physiological functions of water transport across biological membranes have long been the subject of interest. Recent advances in the molecular biology and physiology of water transport have yielded new insights into how and why water moves across cell membranes, and studies on aquaporin knockout mouse models suggest that aquaporins are involved in the development of some diseases and they may be useful targets of research into selective-inhibitor drugs. By focusing on the advances made over the last 30 years in the biophysics, genetics, protein structure, molecular biology, physiology, pathophysiology and pharmacology of aquaporins in mammalian cell membranes, this book provides novel insights into further mechanisms and the physiological significance of water and some small molecule transport in mammals in order to stimulate further research in new directions. In the second version, fourteen chapters will be updated base on the most recent research articles. Ten new chapters will be added.

This book merges approaches in understanding the function of the light-gated ion channels known as channelrhodopsin together with methods addressing how channelrhodopsins can be used to address biomedical questions on a cellular or organismal level. Since the first molecular identification of channelrhodopsins, a broad range of tools have been created and new approaches developed to both better understand the molecular determinants of channelrhodopsin function as well as to use these and homologous proteins from a variety of species as tools to better understand physiological processes, which this volume addresses. Additionally, channelrhodopsins have become instrumental as a potential treatment for disease states. 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, Channelrhodopsin: Methods and Protocols provides a resource for those interested in honing their current expertise in this vital area of study as well as potentially branching out into new directions.

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

Main Question: G protein coupled receptors are involved in highly efficient and specific activation of signalling pathways. How do GPCR signalling complexes get assembled to generate such specificity? In order to answer this question, we need to understand how receptors and their signalling partners are synthesized, folded and quality-controlled in order to generate functional proteins. Then, we need to understand how each partner of the signalling complex is selected to join a complex, and what makes this assembly possible. GPCRs are known to be able to function as oligomers, what drives the assembly into oligomers and what will be the effects of such organization on specificity and efficacy of signal transduction. Once the receptor complexes are assembled, they need to reach different locations in the cell; what drives and controls the trafficking of GPCR signalling complexes. Finally, defects in synthesis, maturation or trafficking can alter functionality of GPCRs signalling complexes; how can we manipulate the system to make it function normally again? Pharmacological chaperones may just be part of the answer to this question.

Christopher M. Cheatum and Amnon Kohen, Relationship of Femtosecond-Picosecond Dynamics to Enzyme-Catalyzed H-Transfer. Cindy Schulenburg and Donald Hilvert, Protein Conformational Disorder and Enzyme Catalysis. A. Joshua Wand, Veronica R. Moorman and Kyle W. Harpole, A Surprising Role for Conformational Entropy in Protein Function. Travis P. Schrank, James O. Wrabl and Vincent J. Hilser, Conformational Heterogeneity Within the LID Domain Mediates Substrate Binding to Escherichia coli Adenylate Kinase: Function Follows Fluctuations. Buyong Ma and Ruth Nussinov, Structured Crowding and Its Effects on Enzyme Catalysis. Michael D. Daily, Haibo Yu, George N. Phillips Jr and Qiang Cui, Allosteric Activation Transitions in Enzymes and Biomolecular Motors: Insights from Atomistic and Coarse-Grained Simulations. Karunesh Arora and Charles L. Brooks III, Multiple Intermediates, Diverse Conformations, and Cooperative Conformational Changes Underlie the Catalytic Hydride Transfer Reaction of Dihydrofolate Reductase. Steven D. Schwartz, Protein Dynamics and the Enzymatic Reaction Coordinate.

This is the second edition of a very well received book that details how the sumoylation system functions and how it modulates numerous cellular activities. SUMO is a post-translational modifier in the ubiquitin super-family that has gained recognition over the last twenty years as an essential and prevalent regulatory molecule. Individual chapters explore the biochemistry, molecular biology, and cell biology of the sumoylation system and its substrate proteins. The book is divided into three themed parts: Molecular Functions (I), Cell Growth Regulation (II), and Diseases (III). Parts I and II focus on the contribution of sumoylation to cellular activities in both the nuclear and cytoplasmic compartments. The nuclear activities covered include nucleic acid metabolism (both RNA and DNA), chromosome structure and replication, and nucleocytoplasmic transport. Cytoplasmic processes presented include regulation of membrane ion channels, general metabolism, and apoptotic signalling. Topics in Part III include the role of sumoylation in developmental abnormalities (craniofacial and cardiovascular), diabetes, neurodegenerative diseases, cancer, and infections with viruses and bacteria. Each of the corresponding chapter authors is an active researcher who has made significant contributions to understanding sumoylation. This second edition provides updates and revisions to most of the original chapters plus adds six new chapters to address important developing areas of sumoylation research. This volume is intended for a scientific audience from undergraduates to independent researchers. The content will serve as both a solid introduction for the novice reader and an in depth treatment for the advanced scholar.

This book focuses on the study of how the properties of nanodiscs, such as lipid composition and size, influence the function of the embedding integral membrane protein, bacteriorhodopsin. The author performed systematic studies to show that the lipid composition and the charge of the hydrophobic head and the structure of hydrophilic tails affect the photocycle pathway of bacteriorhodopsin, which is closely associated with its proton-pumping activity. Furthermore, the author demonstrated a highly efficient method for extracting membrane proteins directly from the biological membrane, preserving protein conformation, function and essential native lipids. This book demonstrates optimization and sample preparation, and presents practical methods of preparing membrane protein-embedded nanodisc samples for biophysical studies, which benefit structural and functional studies in the field of membrane protein characterization, both.

This book discusses a broad range of basic and advanced topics in the field of protein structure, function, folding, flexibility, and dynamics. Starting with a basic introduction to protein purification, estimation, storage, and its effect on the protein structure, function, and dynamics, it also discusses various experimental and computational structure determination approaches; the importance of molecular interactions and water in protein stability, folding and dynamics; kinetic and thermodynamic parameters associated with protein-ligand binding; single molecule techniques and their applications in studying protein folding and aggregation; protein quality control; the role of amino acid sequence in protein aggregation; muscarinic acetylcholine receptors, antimuscarinic drugs, and their clinical significances. Further, the book explains the current understanding on the therapeutic importance of the enzyme dopamine beta hydroxylase; structural dynamics and motions in molecular motors; role of cathepsins in controlling degradation of extracellular matrix during disease states; and the important structure-function relationship of iron-binding proteins, ferritins. Overall, the book is an important guide and a comprehensive resource for understanding protein structure, function, dynamics, and interaction.

This volume explores strategies and detailed protocols for the preparation of macromolecular complexes and their characterization in view of structural analysis. The chapters in this book are separated into three parts: Part One focuses on sample preparation, and covers strategies for recombinant expression of multiprotein complexes in prokaryotic and eukaryotic hosts, for genome engineering using the CRISPR/Cas9 system and for production of specific binders such as reformatted antibodies and artificial binding proteins. Part Two looks at the biophysical methods that can provide useful indicators for sample optimization, and often complement structural information obtained with core technologies for structure determination-x-ray crystallography and cryo-electron microscopy-by quantitative solution data. Part Three discusses the characterization of multiprotein complexes in a cellular environment using the latest technologies and in vivo approaches. 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 authoritative, Multiprotein Complexes: Methods and Protocols is a valuable resource for structural and molecular biologists who need to prepare multi-components for their applications, and for other scientists working on macromolecular assemblies from other angles that need to know the latest approaches that the field has to offer.