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Protein Aggregation In Bacteria
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Book Synopsis Protein Aggregation in Bacteria by : Silvia Maria Doglia
Download or read book Protein Aggregation in Bacteria written by Silvia Maria Doglia and published by John Wiley & Sons. This book was released on 2014-04-03 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: Focuses on the aggregation of recombinant proteins in bacterial cells in the form of inclusion bodies—and on their use in biotechnological and medical applications The first book devoted specifically to the topic of aggregation in bacteria, Protein Aggregation in Bacteria: Functional and Structural Properties of Inclusion Bodies in Bacterial Cells provides a large overview of protein folding and aggregation, including cell biology and methodological aspects. It summarizes, for the first time in one book, ideas and technical approaches that pave the way for a direct use of inclusion bodies in biotechnological and medical applications. Protein Aggregation in Bacteria covers: Molecular and cellular mechanisms of protein folding, aggregation, and disaggregation in bacteria Physiological importance and consequences of aggregation for the bacterial cell Factors inherent to the protein sequence responsible for aggregation and evolutionary mechanisms to keep proteins soluble Structural properties of proteins expressed as soluble aggregates and as inclusion bodies within bacterial cells both from a methodological point of view and with regard to their similarity with amyloids Control of the structural and functional properties of aggregated proteins and use thereof in biotechnology and medicine Protein Aggregation in Bacteria is ideal for researchers in protein science, biochemistry, bioengineering, biophysics, microbiology, medicine, and biotechnology, particularly if they are related with the production of recombinant proteins and pharmaceutical science.
Book Synopsis Protein Solubility and Aggregation in Bacteria by : Salvador Ventura
Download or read book Protein Solubility and Aggregation in Bacteria written by Salvador Ventura and published by Frontiers Media SA. This book was released on 2016-09-26 with total page 129 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins suffer many conformational changes and interactions through their life, from their synthesis at ribosomes to their controlled degradation. Only folded and soluble proteins are functional. Thus, protein folding and solubility are controlled genetically, transcriptionally, and at the protein sequence level. In addition, a well-conserved cellular machinery assists the folding of polypeptides to avoid misfolding and ensure the attainment of soluble and functional structures. When these redundant protective strategies are overcome, misfolded proteins are recruited into aggregates. Recombinant protein production is an essential tool for the biotechnology industry and also supports expanding areas of basic and biomedical research, including structural genomics and proteomics. Although bacteria still represent a convenient production system, many recombinant polypeptides produced in prokaryotic hosts undergo irregular or incomplete folding processes that usually result in their accumulation as insoluble aggregates, narrowing thus the spectrum of protein-based drugs that are available in the biotechnology market. In fact, the solubility of bacterially produced proteins is of major concern in production processes, and many orthogonal strategies have been exploited to try to increase soluble protein yields. Importantly, contrary to the usual assumption that the bacterial aggregates formed during protein production are totally inactive, the presence of a fraction of molecules in a native-like structure in these assemblies endorse them with a certain degree of biological activity, a property that is allowing the use of bacteria as factories to produce new functional materials and catalysts. The protein embedded in intracellular bacterial deposits might display different conformations, but they are usually enriched in beta-sheet-rich assemblies resembling the amyloid fibrils characteristic of several human neurodegenerative diseases. This makes bacterial cells simple, but biologically relevant model systems to address the mechanisms behind amyloid formation and the cellular impact of protein aggregates. Interestingly, bacteria also exploit the structural principles behind amyloid formation for functional purposes such as adhesion or cytotoxicity. In the present research topic we collect papers addressing all the issues mentioned above from both the experimental and computational point of view.
Book Synopsis Protein Solubility and Aggregation in Bacteria by :
Download or read book Protein Solubility and Aggregation in Bacteria written by and published by . This book was released on 2016 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins suffer many conformational changes and interactions through their life, from their synthesis at ribosomes to their controlled degradation. Only folded and soluble proteins are functional. Thus, protein folding and solubility are controlled genetically, transcriptionally, and at the protein sequence level. In addition, a well-conserved cellular machinery assists the folding of polypeptides to avoid misfolding and ensure the attainment of soluble and functional structures. When these redundant protective strategies are overcome, misfolded proteins are recruited into aggregates. Recombinant protein production is an essential tool for the biotechnology industry and also supports expanding areas of basic and biomedical research, including structural genomics and proteomics. Although bacteria still represent a convenient production system, many recombinant polypeptides produced in prokaryotic hosts undergo irregular or incomplete folding processes that usually result in their accumulation as insoluble aggregates, narrowing thus the spectrum of protein-based drugs that are available in the biotechnology market. In fact, the solubility of bacterially produced proteins is of major concern in production processes, and many orthogonal strategies have been exploited to try to increase soluble protein yields. Importantly, contrary to the usual assumption that the bacterial aggregates formed during protein production are totally inactive, the presence of a fraction of molecules in a native-like structure in these assemblies endorse them with a certain degree of biological activity, a property that is allowing the use of bacteria as factories to produce new functional materials and catalysts. The protein embedded in intracellular bacterial deposits might display different conformations, but they are usually enriched in beta-sheet-rich assemblies resembling the amyloid fibrils characteristic of several human neurodegenerative diseases. This makes bacterial cells simple, but biologically relevant model systems to address the mechanisms behind amyloid formation and the cellular impact of protein aggregates. Interestingly, bacteria also exploit the structural principles behind amyloid formation for functional purposes such as adhesion or cytotoxicity. In the present research topic we collect papers addressing all the issues mentioned above from both the experimental and computational point of view.
Book Synopsis Functions and Mechanisms of Bacterial Protein Homeostasis and Stress Responses by : Axel Mogk
Download or read book Functions and Mechanisms of Bacterial Protein Homeostasis and Stress Responses written by Axel Mogk and published by Frontiers Media SA. This book was released on 2022-02-01 with total page 334 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Cover Image for This Research Topic is Used With Permission of the Authors and Publishers of the Following Article: Winkler J, Seybert A, König L, Pruggnaller S, Haselmann U, Sourjik V, Weiss M, Frangakis AS, Mogk A, Bukau B.EMBO J. 2010 Mar 3;29(5):910-23. doi: 10.1038/emboj.2009.412. Epub 2010 Jan 21
Book Synopsis Protein Aggregation by : Andrzej Stanisław Cieplak
Download or read book Protein Aggregation written by Andrzej Stanisław Cieplak and published by Springer Nature. This book was released on 2022-10-30 with total page 673 pages. Available in PDF, EPUB and Kindle. Book excerpt: The volume details techniques, methods, and conceptual developments to further the study of protein aggregation with emphasis on the pleiomorphic proteins implicated in etiology of neurodegeneration. Chapters guide readers through in vitro and in vivo studies of fibrillization and liquid-liquid phase separation processes, and offer a comprehensive account of the state-of-art of structural studies of protein aggregation. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Protein Aggregation: Methods and Protocols aims to be useful and practical guide to new researchers and experts looking to expand their knowledge.
Book Synopsis Protein Secretion in Bacteria by : Maria Sandkvist
Download or read book Protein Secretion in Bacteria written by Maria Sandkvist and published by John Wiley & Sons. This book was released on 2019-09-01 with total page 626 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein transport into and across membranes is a fundamental process in bacteria that touches upon and unites many areas of microbiology, including bacterial cell physiology, adhesion and motility, nutrient scavenging, intrabacterial signaling and social behavior, toxin deployment, interbacterial antagonism and collaboration, host invasion and disruption, and immune evasion. A broad repertoire of mechanisms and macromolecular machines are required to deliver protein substrates across bacterial cell membranes for intended effects. Some machines are common to most, if not all bacteria, whereas others are specific to Gram-negative or Gram-positive species or species with unique cell envelope properties such as members of Actinobacteria and Spirochetes. Protein Secretion in Bacteria, authored and edited by an international team of experts, draws together the many distinct functions and mechanisms involved in protein translocation in one concise tome. This comprehensive book presents updated information on all aspects of bacterial protein secretion encompassing: Individual secretory systems–Sec, Tat, and T1SS through the newly discovered T9SS Mechanisms, structures, and functions of bacterial secretion systems Lipoprotein sorting pathways, outer membrane vesicles, and the sortase system Structures and roles of surface organelles, including flagella, pili, and curli Emerging technologies and translational implications Protein Secretion in Bacteria serves as both an introductory guide for students and postdocs and a ready reference for seasoned researchers whose work touches on protein export and secretion. This volume synthesizes the diversity of mechanisms of bacterial secretion across the microbial world into a digestible resource to stimulate new research, inspire continued identification and characterization of novel systems, and bring about new ways to manipulate these systems for biotechnological, preventative, and therapeutic applications.
Book Synopsis Protein Secretion Pathways in Bacteria by : B. Oudega
Download or read book Protein Secretion Pathways in Bacteria written by B. Oudega and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 404 pages. Available in PDF, EPUB and Kindle. Book excerpt: For bacteria ..."the times are achanging"... The genomes of over 60 different bacteria have now been sequenced, and we know a lot about the important research organism Escherichia coli, the important industrial organism Bacillus subtilis, and about important plant and human pathogens. It will not take long before we know all the gene products and their functions of a few of these bacteria. Some of us already begin to think about a digital model E. coli or Bacillus cell. For that end we need to know all the physiological activities and metabolic routes of the cell. But in addition we like to know how things work at the molecular level and how protein and membranes as well as other (macromolecular) structures work together to carry out specific cell functions. Protein Secretion Pathways in Bacteria describes all the known folding and targeting routes of inner and outer membrane proteins as well as of proteins that are secreted by several specific export routes. The book gives detailed molecular information about the structures that are important for the different mechanisms involved. This is a valuable contribution to the understanding of how rather simple and yet complex bacterial cells work.
Book Synopsis Protein Aggregation by : Douglas A. Stein
Download or read book Protein Aggregation written by Douglas A. Stein and published by Nova Biomedical Books. This book was released on 2011 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein aggregation is the aggregation of mis-folded proteins, and is thought to be responsible for many degenerative diseases, such as Alzheimer's. This book presents current research from across the globe in the study of protein aggregation, including the processes of protein aggregation induced by freezing and lyophilization; functional amyloids; thermally induced aggregation of a model system protein - insulin; the aggregation of albumin; synucleins implicated in neurodegenerative diseases and some forms of cancer; yeast protein aggregates; and the folding and aggregation features of proteins.
Download or read book Microbial Aggregation written by Calleja and published by CRC Press. This book was released on 2018-05-04 with total page 434 pages. Available in PDF, EPUB and Kindle. Book excerpt: This text covers in detail bacteria and yeasts, including an overall perspective of microbial aggregation as fundamental form and function, which is presented here to include systems still to be treated in detail.
Book Synopsis Recombinant protein expression in microbial systems by : Eduardo A. Ceccarelli
Download or read book Recombinant protein expression in microbial systems written by Eduardo A. Ceccarelli and published by Frontiers E-books. This book was released on 2014-10-02 with total page 103 pages. Available in PDF, EPUB and Kindle. Book excerpt: With the advent of recombinant DNA technology, expressing heterologous proteins in microorganisms rapidly became the method of choice for their production at laboratory and industrial scale. Bacteria, yeasts and other hosts can be grown to high biomass levels efficiently and inexpensively. Obtaining high yields of recombinant proteins from this material was only feasible thanks to constant research on microbial genetics and physiology that led to novel strains, plasmids and cultivation strategies. Despite the spectacular expansion of the field, there is still much room for progress. Improving the levels of expression and the solubility of a recombinant protein can be quite challenging. Accumulation of the product in the cell can lead to stress responses which affect cell growth. Buildup of insoluble and biologically inactive aggregates (inclusion bodies) lowers the yield of production. This is particularly true for obtaining membrane proteins or high-molecular weight and multi-domain proteins. Also, obtaining eukaryotic proteins in a prokaryotic background (for example, plant or animal proteins in bacteria) results in a product that lack post-translational modifications, often required for functionality. Changing to a eukaryotic host (yeasts or filamentous fungi) may not be a proper solution since the pattern of sugar modifications is different than in higher eukaryotes. Still, many advances in the last couple of decades have provided to researchers a wide variety of strategies to maximize the production of their recombinant protein of choice. Everything starts with the careful selection of the host. Be it bacteria or yeast, a broad list of strains is available for overcoming codon use bias, incorrect disulfide bond formation, protein toxicity and lack of post-translational modifications. Also, a huge catalog of plasmids allows choosing for different fusion partners for improving solubility, protein secretion, chaperone co-expression, antibiotic resistance and promoter strength. Next, controlling culture conditions like temperature, inducer and media composition can bolster recombinant protein production. With this Research Topic, we aim to provide an encyclopedic account of the existing approaches to the expression of recombinant proteins in microorganisms, highlight recent discoveries and analyze the future prospects of this exciting and ever-growing field.
Book Synopsis Protein Aggregation in Biological Cells by : Stuart Joseph Rubin
Download or read book Protein Aggregation in Biological Cells written by Stuart Joseph Rubin and published by . This book was released on 1983 with total page 82 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Contribution of the Ribosome and Molecular Chaperones to [de Novo] Protein Folding in Bacteria by : Rayna Addabbo
Download or read book Contribution of the Ribosome and Molecular Chaperones to [de Novo] Protein Folding in Bacteria written by Rayna Addabbo and published by . This book was released on 2018 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein folding is the critical process by which proteins achieve their native conformations, often necessary to perform life-sustaining functions for the cell. While all organisms rely on protein folding for survival, it is not well understood how proteins fold, especially within the complex environment of the cell. While evolution has optimized protein folding for success, it is not an infallible process. Aside from the many human diseases whose pathology is closely linked to protein misfolding and aggregation, aberrant folding has made it challenging for researchers to use recombinant DNA technologies to over-produce soluble functional proteins. This limitation has had significant consequences for research, biotechnology and medicine, particularly in the field of protein-based biopharmaceuticals. It follows that there is great motivation to understand how cells successfully produce structurally accurate proteins. In this thesis I will gain insights into the parameters that dictate whether a protein successfully folds or aggregates upon release from the ribosome. I will then examine how molecular chaperones promote protein folding and the prevention of protein aggregation. This thesis is divided into four chapters. Chapter 1 is an introductory chapter that reviews the current literature on protein folding in the cell and incorporates my current research findings. This chapter aims at highlighting why proteins need the cellular environment for successful folding and prevention of aggregation. In Chapter 2, I explore how co-and-posttranslational protein folding in the absence of molecular chaperones is related to successful native state formation and to detrimental aggregation. I describe investigations showing that the ribosome plays a critical role in promoting nascent-protein solubility during biosynthesis. Further, I describe the features of an important kinetic competition that takes place upon protein release from the ribosome immediately following translation. During this time, proteins are irreversibly channeled towards either their native state or aggregated conformations. I find that, to avoid aggregation, proteins must be able to fold faster than they aggregate, as they depart from the environment established by the ribosomal surface and its immediate vicinity. Before completion of translation, the ribosomal environment discourages aggregation due to the large size of the ribosome-nascent-protein complex (RNC). However, this translational-diffusion advantage vanishes after the newly synthesized protein departs from the ribosome. In summary, this chapter highlights the role of the ribosome in promoting the solubility of newly synthesized proteins. The sequence and structural diversity of proteins in the E. coli cytosol implies that not all proteins have the same ability to fold rapidly upon release from the ribosome. In Chapters 3 and 4, I focus on understanding how the highly conserved molecular chaperone Hsp70 mediates protein folding and helps preventing aggregation in bacteria. In Chapter 3, I experimentally demonstrate that the Hsp70 molecular chaperone (also known as DnaK in bacteria) promotes protein structural accuracy and native-state formation. When proteins are biosynthesized at sub-optimal Hsp70 concentration, a mixture of the native state and aggregated species is produced. Surprisingly, even proteins that are soluble after release from the ribosome in the absence of Hsp70 are not necessarily in their native conformation. These proteins often include a significant population of soluble aggregate as well as soluble native state. In summary, this chapter highlights the key role of the Hsp70 molecular chaperone for protein structural accuracy upon release from the ribosome. In Chapter 4, I employ a combination of experiments and computation to show that the Hsp70 chaperone concentration and the chaperone-to-protein ratio needed for protein solubility and structural accuracy are directly dependent on the features of the kinetic partitioning as nascent proteins are release from the ribosome (see Chapter 2). Proteins that aggregate rapidly relative to folding are more highly dependent upon interactions with DnaK upon release from the ribosome. It follows that Hsp70-mediated protein folding promotes the existence of a more diverse cellular proteome by allowing the cell to access slow-folding and aggregation-prone proteins that would otherwise not be accessible. The findings in this dissertation are consistent with a global view of cellular protein folding according to which folding begins on the ribosome. The role of the ribosome is to support conformational sampling and solubility while molecular chaperones ensure structural accuracy as well as high yields of native state. Depending upon the folding and aggregation properties of the protein under investigation, different proteins rely on the ribosome and molecular chaperones to a different extent, for successful protein folding. The dual dependence on the ribosome and molecular chaperones provides a robust machinery to produce a variety of proteins needed to support life and properly promote sequence, structural and functional diversity.
Book Synopsis Bacterial Persistence by : Jan Michiels
Download or read book Bacterial Persistence written by Jan Michiels and published by Humana. This book was released on 2015-10-15 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume presents a comprehensive collection of methods that have been instrumental to the current understanding of bacterial persisters. Chapters in the book cover topics ranging from general methods for measuring persister levels in Escherichia coli cultures, protocols for the determination of the persister subpopulation in Candida albicans, quantitative measurements of Type I and Type II persisters using ScanLag, to in vitro and in vivo models for the study of the intracellular activity of antibiotics. 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, Bacterial Persistence: Methods and Protocols brings together the most respected researchers in bacterial persistence whose studies will remain vital to understanding this field for many years to come.
Book Synopsis Protein Homeostasis in Survival and Persistence of Escherichia Coli, Salmonella Typhimurium and Cronobacter Sakazakii at Alkaline PH and After Desiccation by : Tongbo Zhu
Download or read book Protein Homeostasis in Survival and Persistence of Escherichia Coli, Salmonella Typhimurium and Cronobacter Sakazakii at Alkaline PH and After Desiccation written by Tongbo Zhu and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacteria have evolved a protein homeostasis network to maintain the integrity of their proteome. Chaperones and proteases of the core genome can be complemented by accessory genes encoding additional elements of the protein homeostasis network. The transmissible locus of stress tolerance (tLST) confers exceptional tolerance to high temperature, chlorine, oxidative chemicals and high hydrostatic pressure by reducing protein aggregation. Highly expressed tLST proteins include intracellular small heat shock proteins sHsp20 and sHspGI, disaggregase ClpKGI, and periplasmic stress chaperones PscA and PscB. The tLST also encodes KefBGI, a Na+/H+ antiporter. This study aimed to determine the effects of improved protein homeostasis that is mediated by the tLST on bacterial tolerance to alkaline pH and desiccation. The expression of the Cpx, a two-component regulatory system in Escherichia coli which responds to envelope stress, was not altered by the presence of the tLST. The tolerance or resistance of E. coli to alkaline pH in the pH range of 6.9 to 9.2 and at pH 11, respectively, were also not changed by the presence of the tLST. The presence of the tLST improved, however, survival at pH 11 in presence of chlorine stress; this effect was attributed to KefBGI rather than protein homeostasis. The impact of protein homeostasis on desiccation tolerance was characterized in Salmonella enterica serovar Typhimurium, Cronobacter sakazakii and E. coli. The cloning and expression of the shsp20, shspGI and clpKGI decreased desiccation tolerance in S. Typhimurium and C. sakazakii but not in E. coli. Protein aggregates were visualized in vivo using an IbpA-Yfp fusion protein, demonstrating that cloning of shsp20, shspGI and clpKGI reduced intracellular protein aggregates in S. Typhimurium and C. sakazakii but not in E. coli. The presence of the elements intensively consuming cellular resources including high copy plasmids as well as cloning of highly expressed proteins had a detrimental effect on desiccation tolerance irrespective of the function of the expressed proteins. Abolishing the ATP-hydrolysis function of ClpKGI by substitution of two amino acids (E383A/E723A) increased cell counts of S. Typhimurium after desiccation more than 100-fold, demonstrating a direct contribution of protein aggregation to desiccation tolerance. Dry storage of S. Typhimurium and C. sakazakii for 112 days revealed that cloning of the tLST or of its protein homeostasis module decreased survival during desiccated storage in infant formula. In conclusion, additional protein quality control provided by tLST does not aid in alkaline resistance or tolerance in E. coli. The desiccation tolerance of S. Typhimurium and C. sakazakii positively correlates with the formation of protein aggregates but are negatively impacted by the presence of the elements intensively consuming cellular resources. Taken together, this study improves the understanding of how protein homeostasis affects bacterial stress tolerance by increasing tolerance to some stressors while reducing tolerance to others.
Book Synopsis Inclusions in Prokaryotes by : Jessup M. Shively
Download or read book Inclusions in Prokaryotes written by Jessup M. Shively and published by Springer Science & Business Media. This book was released on 2006-05-04 with total page 353 pages. Available in PDF, EPUB and Kindle. Book excerpt: The new series "Microbiology Monographs" begins with two volumes on intracellular components in prokaryotes. In this first volume, "Inclusions in Prokaryotes", the components, labeled inclusions, are defined as discrete bodies resulting from synthesis of a metabolic product. Research on the biosynthesis and reutilization of the accumulated materials is still in progress, and interest in the inclusions is growing. This comprehensive volume provides historical background and comprehensive reviews of eight well-known prokaryotic inclusions.
Book Synopsis Protein Folding and Aggregation in the Presence of the Hsp70 Chaperone by : Miranda F. Mecha
Download or read book Protein Folding and Aggregation in the Presence of the Hsp70 Chaperone written by Miranda F. Mecha and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Most life on earth depends on ribosome-assisted biosynthesis and on the generation and preservation of correct protein structure. Molecular chaperones and their cochaperones act co- and post-translationally to promote de novo protein folding, overcome protein damage upon stress and even disaggregate protein aggregates. Hsp70, a ubiquitous and highly conserved 70 kDa heat shock protein, is a particularly important and well-studied chaperone. It is often referred to as a central "hub" due to its myriad of functions and its profound effect on cell viability. While the Hsp70 chaperone cycle has been well-documented in the literature, there is still much to be understood about the interplay between Hsp70 and its client-proteins, including the kinetic and thermodynamic client-protein characteristics required for interaction with Hsp70. The Hsp70 chaperone is nucleotide-dependent and derives part of its driving force for assisting protein folding from ATP hydrolysis. The Hsp70-related studies carried out to date bear an apparent inconsistency. Namely, some proteins were reported to attain their native state more slowly in the presence of the Hsp70 chaperone than under chaperone-free conditions. On the other hand, aggregation-prone proteins routinely acquire a bioactive native state faster, in the presence of Hsp70. Part of the work carried out in this thesis attempts to explain this apparent inconsistency. In addition, we explore the kinetic and thermodynamic client-protein characteristics necessary for interaction with the Hsp70 chaperone. Finally, we address the relation between protein aging and Hsp70-chaperone activity.The thesis is divided into six chapters. Chapter 1 delves into the current literature and summarizes what is known about protein folding and how the folding process is influenced by the Hsp70 chaperone cycle. This chapter further discusses the structure and function of Hsp70 and how these characteristics affect the conformation and dynamics of chaperone-bound client proteins. The chapter also provides a brief overview of the current computational approaches to predict the timecourse of Hsp70-assisted protein folding. Chapter 2 focuses on the development of CHAMPION70, a computational model able to perform Chaperone-Mediated Protein folding kinetic Simulations involving Hsp70. We then apply CHAMPION70 to four classes of client proteins with different kinetic (fast- or slow-folding) and thermodynamic (stable or unstable) stabilities in the presence of either no aggregation, weak aggregation or strong aggregation propensities. We find that, in the absence of aggregation, unstable client proteins capture (i.e., stay bound to) the Hsp70 chaperone indefinitely. This is a clear disadvantage unless Hsp70 serves as a transport machine, for these proteins. Conversely, in the presence of weak or strong aggregation propensities, it is very beneficial for client-proteins to interact with the Hsp70 chaperone system. Specifically, slow-folding and thermodynamically stable client proteins experience the greatest aggregation-prevention advantages in the presence of Hsp70, especially if the class of client proteins is strongly aggregation-prone. However, Hsp70 is unable to assist the folding of strongly aggregation-prone and thermodynamically unstable proteins. Importantly, we also predict that the E. coli Hsp70 chaperone system is unable to prevent protein aggregation over long time spans long-term (i.e., greater than ca 60 years). This result suggests that one of the consequence of protein aging is the intrinsic failure of the bacterial Hsp70 chaperone machinery. Of course E. coli bacteria double in only a few minutes and "old proteins" likely persist in the progeny (i.e., daughter cells). Yet these old proteins progressively become more and more dilute, hence less-aggregation-prone. This phenomenon may rescue bacteria from disaster. Yet one wonders whether this effect may have a more severe impact on eukaryotic Hsp70s. In summary, the CHAMPION70 simulator is a powerful tool to enable the prediction of client-protein behavior in the presence of one of the most amazing cellular machines, the Hsp70 chaperone system. Chapter 3 provides simple computational tools to discriminate folded from intrinsically disordered proteins (IDPs) under physiologically relevant conditions, solely based on protein amino-acid composition. This tool only requires knowledge on protein hydrophobicity-per-residue and net-charge-per-residue. The net-charge-non-polar (NECNOP) algorithm results in 95% accuracy, and this value increases for proteins of more than 140 residues. Chapter 4 delves into influence of the E. coli ribosome on both co- and post-translational protein folding in the absence typical molecular chaperone systems (DnaK, trigger factor) and in the presence of aggregation. In this experimental investigation, translation through the ribosome is found to promote nascent-protein solubility even in the absence of cotranslationally active molecular chaperones. This work also shows that the E. coli trigger factor and DnaK molecular chaperones increase the solubility of nascent chains emerging from the ribosomal exit tunnel and minimize co- and post-translational aggregation. Most importantly, this work shows the importance of immediately post-translational kinetic partitioning of nascent proteins between native-state and aggregates, upon release form the ribosome. This partitioning is dramatically sensitive to subtle variations in amino-acid sequence, including single-point mutations. Chapter 5 demonstrates the increased sensitivity of the NMR hyperpolarization technique known as low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP). This technique is used for detection of aromatic amino acids in the presence of both a photosensitizer dye (fluorescein) and a cryogenic probe. Experiments rapidly detect the amino acids tryptophan (Trp) and tyrosine (Tyr) at unprecedented concentrations (200 nM). Detection of the model protein drkN SH3 (which bears Trp, Tyr and His) at 500 nM on a 600 MHz spectrometer via LC-Photo-CIDNP leads to a 30-fold better S/N relative to conventional 2D experiments performed at higher magnetic field (900MHz spectrometer). Spectral editing of the model protein allowed for secondary and tertiary structure analysis. In contrast to regular photo-CIDNP, LC-photo-CIDNP does not heavily depend on laser intensity, thus allowing for safer and more cost-effective experiments. Chapter 6 further develops the investigations of Chapter 5 on LC-photo-CIDNP. A major limitation of LC-photo-CIDNP is that a limited number of scans (up to ca 200) can typically be collected before sample degradation takes over. The signal-to-noise (SN) ratio becomes progressively weaker as the number of scans increases. This disadvantage strongly limits the ability to perform long-term experiments. Two reductive radical quenchers - ascorbic acid (vitamin C) and 2-mercaptoethylamine (MEA) - were employed in this study, to minimize the extent of photodamage in NMR samples. This technique both enhanced the S/N by over 100% and allowed for more transients to be acquired for amino-acid and protein samples in solution.
Book Synopsis Protein Aggregation in Bacteria by : Silvia Maria Doglia
Download or read book Protein Aggregation in Bacteria written by Silvia Maria Doglia and published by Wiley. This book was released on 2014-04-14 with total page 288 pages. Available in PDF, EPUB and Kindle. Book excerpt: Focuses on the aggregation of recombinant proteins in bacterial cells in the form of inclusion bodies—and on their use in biotechnological and medical applications The first book devoted specifically to the topic of aggregation in bacteria, Protein Aggregation in Bacteria: Functional and Structural Properties of Inclusion Bodies in Bacterial Cells provides a large overview of protein folding and aggregation, including cell biology and methodological aspects. It summarizes, for the first time in one book, ideas and technical approaches that pave the way for a direct use of inclusion bodies in biotechnological and medical applications. Protein Aggregation in Bacteria covers: Molecular and cellular mechanisms of protein folding, aggregation, and disaggregation in bacteria Physiological importance and consequences of aggregation for the bacterial cell Factors inherent to the protein sequence responsible for aggregation and evolutionary mechanisms to keep proteins soluble Structural properties of proteins expressed as soluble aggregates and as inclusion bodies within bacterial cells both from a methodological point of view and with regard to their similarity with amyloids Control of the structural and functional properties of aggregated proteins and use thereof in biotechnology and medicine Protein Aggregation in Bacteria is ideal for researchers in protein science, biochemistry, bioengineering, biophysics, microbiology, medicine, and biotechnology, particularly if they are related with the production of recombinant proteins and pharmaceutical science.
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