Download Protein Solubility And Aggregation In Bacteria full books in PDF, epub, and Kindle. Read online Protein Solubility And Aggregation In Bacteria ebook anywhere anytime directly on your device. Fast Download speed and no annoying ads. We cannot guarantee that every ebooks is available!
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 : 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.
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.
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 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 2014-04-14 with total page 0 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.
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 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.
Proteins are responsible for driving a vast majority of biological processes in the cell. Therefore, it is important that proteins remain mostly folded to carry out their important functions, after transcription-translation. Prior proteome-wide studies showed that proteins in the Escherichia coli proteome are at risk of misfolding and extensive aggregation after translation, especially under cellular or environmental stresses. In addition, aggregates have been shown to be more thermodynamically stable than the native states, for many proteins. Therefore, if misfolding and aggregation in the cell were thermodynamically driven processes, many proteins in the E. coli proteome would spontaneously form aggregates in the cell. Despite a plethora of protein quality control and degradation systems employed by E. coli, it is clear that these systems are energetically expensive and can be overwhelmed, especially if many proteins in the proteome are at risk of thermodynamically driven aggregation. One popular proposed concept in the folding field is that the proteome is kinetically protected from aggregation, avoiding significant reliance on ATP-expensive protein quality control and degradation systems, under cellular conditions. However, there have not been experiments directly proving kinetic stability of proteins relative to aggregation on a proteome-wide scale. Moreover, aggregates are initially formed as soluble non-native oligomers, or soluble aggregates. These soluble aggregates may cause impairments of important biological processes in the cell and can eventually form larger insoluble assemblies. Yet, little is known about how soluble aggregates fit in the life cycle of E. coli proteins. To address the above fascinating biological questions, in this thesis, I will explore the kinetic stability and aggregation of a representative E. coli proteome (A19 cell strain). This Ph.D. thesis includes three Chapters. Chapter 1 includes background information on protein folding and aggregation in the cellular context. In addition, it discusses the importance of proteome-wide studies and the important findings on aggregation from current proteomic analyses and introduces the hypothesis of proteome kinetic stability relative to aggregation. This Chapter serves as the conceptual basis for the subsequent Chapters. Chapter 2 explores the proteome-wide kinetic stability of proteins in E. coli under physiologically relevant conditions. Here, I demonstrate that the free-energy landscape of the E. coli proteome includes extensive insoluble aggregates under physiologically relevant conditions. Further, the soluble and insoluble aggregates can exchange reversibly among each other and the apparent critical concentration for soluble-aggregate formation on a proteome-wide scale is very low (c.a., 0.04 [mu]g/mL). I demonstrate that over 80% of the proteome is kinetically protected from forming these soluble and insoluble aggregates on timescales longer than this organism's doubling time. Using bottom-up proteomics, I report that over 800 E. coli proteins are kinetically stable relative to aggregation regardless of structure, function, and cellular location. Finally, my results show that cytoplasmic/periplasmic molecular chaperones are amongst the most soluble proteins, both at higher temperature and under physiologically relevant conditions. This finding demonstrates that molecular chaperones are extremely robust members of the proteome. Chapter 3 focuses on the development of a novel isotopolog of tryptophan to enhance the sensitivity of NMR spectroscopy and enable monitoring protein behaviors in complex environments. Here, a novel selectively isotopic-labeled tryptophan was synthesized and successfully detected at very low concentrations in buffer and in a complex S30 cell extract (c.a., 20 nM and 1 [mu]M respectively) by low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) NMR. Our new selective labeling approach to LC-photo-CIDNP, in combination with existing biophysical analyses, can be utilized to study the effect of heterologous expression. For instance, this Trp isotopolog will enable the monitoring of folding and aggregation of model recombinant proteins within in complex cell-like environments. Overall, I propose that proteome-wide kinetic stability is an effective strategy to maintain a healthy and aggregation-free cellular environment in living systems, without reliance on energetically expensive degradation and disaggregation processes. These results also provide insights into the structural and functional determinants of bacterial kinetic stability and aggregation. In conclusion, it is hoped that the knowledge gained from this work will ultimately benefit the design and discovery of new strategies to prevent protein aggregation in the cell and to improve the shelf life of many protein-based biologics (e.g., monoclonal antibodies, etc).
Book Synopsis Exploring Kinetic Controlled Protein Solubility Under Physiologically Relevant Conditions by : Hung Hoang Dang
Download or read book Exploring Kinetic Controlled Protein Solubility Under Physiologically Relevant Conditions written by Hung Hoang Dang and published by . This book was released on 2024 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are responsible for driving a vast majority of biological processes in the cell. Therefore, it is important that proteins remain mostly folded to carry out their important functions, after transcription-translation. Prior proteome-wide studies showed that proteins in the Escherichia coli proteome are at risk of misfolding and extensive aggregation after translation, especially under cellular or environmental stresses. In addition, aggregates have been shown to be more thermodynamically stable than the native states, for many proteins. Therefore, if misfolding and aggregation in the cell were thermodynamically driven processes, many proteins in the E. coli proteome would spontaneously form aggregates in the cell. Despite a plethora of protein quality control and degradation systems employed by E. coli, it is clear that these systems are energetically expensive and can be overwhelmed, especially if many proteins in the proteome are at risk of thermodynamically driven aggregation. One popular proposed concept in the folding field is that the proteome is kinetically protected from aggregation, avoiding significant reliance on ATP-expensive protein quality control and degradation systems, under cellular conditions. However, there have not been experiments directly proving kinetic stability of proteins relative to aggregation on a proteome-wide scale. Moreover, aggregates are initially formed as soluble non-native oligomers, or soluble aggregates. These soluble aggregates may cause impairments of important biological processes in the cell and can eventually form larger insoluble assemblies. Yet, little is known about how soluble aggregates fit in the life cycle of E. coli proteins. To address the above fascinating biological questions, in this thesis, I will explore the kinetic stability and aggregation of a representative E. coli proteome (A19 cell strain). This Ph.D. thesis includes three Chapters. Chapter 1 includes background information on protein folding and aggregation in the cellular context. In addition, it discusses the importance of proteome-wide studies and the important findings on aggregation from current proteomic analyses and introduces the hypothesis of proteome kinetic stability relative to aggregation. This Chapter serves as the conceptual basis for the subsequent Chapters. Chapter 2 explores the proteome-wide kinetic stability of proteins in E. coli under physiologically relevant conditions. Here, I demonstrate that the free-energy landscape of the E. coli proteome includes extensive insoluble aggregates under physiologically relevant conditions. Further, the soluble and insoluble aggregates can exchange reversibly among each other and the apparent critical concentration for soluble-aggregate formation on a proteome-wide scale is very low (c.a., 0.04 [mu]g/mL). I demonstrate that over 80% of the proteome is kinetically protected from forming these soluble and insoluble aggregates on timescales longer than this organism's doubling time. Using bottom-up proteomics, I report that over 800 E. coli proteins are kinetically stable relative to aggregation regardless of structure, function, and cellular location. Finally, my results show that cytoplasmic/periplasmic molecular chaperones are amongst the most soluble proteins, both at higher temperature and under physiologically relevant conditions. This finding demonstrates that molecular chaperones are extremely robust members of the proteome. Chapter 3 focuses on the development of a novel isotopolog of tryptophan to enhance the sensitivity of NMR spectroscopy and enable monitoring protein behaviors in complex environments. Here, a novel selectively isotopic-labeled tryptophan was synthesized and successfully detected at very low concentrations in buffer and in a complex S30 cell extract (c.a., 20 nM and 1 [mu]M respectively) by low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) NMR. Our new selective labeling approach to LC-photo-CIDNP, in combination with existing biophysical analyses, can be utilized to study the effect of heterologous expression. For instance, this Trp isotopolog will enable the monitoring of folding and aggregation of model recombinant proteins within in complex cell-like environments. Overall, I propose that proteome-wide kinetic stability is an effective strategy to maintain a healthy and aggregation-free cellular environment in living systems, without reliance on energetically expensive degradation and disaggregation processes. These results also provide insights into the structural and functional determinants of bacterial kinetic stability and aggregation. In conclusion, it is hoped that the knowledge gained from this work will ultimately benefit the design and discovery of new strategies to prevent protein aggregation in the cell and to improve the shelf life of many protein-based biologics (e.g., monoclonal antibodies, etc).
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 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.
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 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.
Despite its fundamental importance for life, many details regarding how the cell promotes the solubility and structural accuracy of proteins remains poorly understood. This lack of knowledge poses serious challenges in basic science, biotechnology and medicine as the inability to prevent inclusion body formation limits the efficient overproduction of recombinant proteins and protein-based therapeutics. In this thesis, experimental and computational approaches were combined to explore factors that help discriminate folding and aggregation pathways at various stages of a protein's life in the cell. If folded properly, the native states of many proteins, including apomyoglobin and the soluble E. coli proteome, were shown to be kinetically-trapped from stable aggregate states under physiologically-relevant conditions. However, kinetic simulations suggest that this kinetic trapping can be circumvented at high protein concentrations and, importantly, in the presence of small pre-nucleated aggregate seeds capable of further elongation. In order to further explore how the intracellular environment further influences protein folding and aggregation, we developed a novel bacterial Hsp70 inhibitor which targets and inhibits DnaK. This inhibitor is functional in dilute buffer and cell-free systems. Importantly, it does not also inhibit nascent protein biosynthesis. Our novel inhibitor was then used to probe how the ribosome and DnaK coordinate co- and post-translational events which ultimately support proper protein folding. The ribosome was observed to act as a powerful facilitator of protein solubility during the earliest stages of translation and upon ribosome release. However, it requires additional help from molecular chaperones (e.g. DnaK) to ensure the soluble proteins produced are structurally accurate. Kinetic simulations further highlight that this chaperone requirement for proper folding increases for proteins which are slow to fold at the end of translation. This highlights the synergistic, yet distinct, roles of the ribosome and DnaK required to produce soluble, properly folded proteins which would otherwise be aggregation-prone on their own. Taken together, the work highlighted in this thesis sheds light on the unique mechanism by which the cell shapes, and ultimately traps, proteins into their native state during the earliest stages of protein life.
Book Synopsis The Earliest Stages of a Protein's Life Influence Its Long-term Solubility and Structural Accuracy by : Matthew Dalphin
Download or read book The Earliest Stages of a Protein's Life Influence Its Long-term Solubility and Structural Accuracy written by Matthew Dalphin and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Despite its fundamental importance for life, many details regarding how the cell promotes the solubility and structural accuracy of proteins remains poorly understood. This lack of knowledge poses serious challenges in basic science, biotechnology and medicine as the inability to prevent inclusion body formation limits the efficient overproduction of recombinant proteins and protein-based therapeutics. In this thesis, experimental and computational approaches were combined to explore factors that help discriminate folding and aggregation pathways at various stages of a protein's life in the cell. If folded properly, the native states of many proteins, including apomyoglobin and the soluble E. coli proteome, were shown to be kinetically-trapped from stable aggregate states under physiologically-relevant conditions. However, kinetic simulations suggest that this kinetic trapping can be circumvented at high protein concentrations and, importantly, in the presence of small pre-nucleated aggregate seeds capable of further elongation. In order to further explore how the intracellular environment further influences protein folding and aggregation, we developed a novel bacterial Hsp70 inhibitor which targets and inhibits DnaK. This inhibitor is functional in dilute buffer and cell-free systems. Importantly, it does not also inhibit nascent protein biosynthesis. Our novel inhibitor was then used to probe how the ribosome and DnaK coordinate co- and post-translational events which ultimately support proper protein folding. The ribosome was observed to act as a powerful facilitator of protein solubility during the earliest stages of translation and upon ribosome release. However, it requires additional help from molecular chaperones (e.g. DnaK) to ensure the soluble proteins produced are structurally accurate. Kinetic simulations further highlight that this chaperone requirement for proper folding increases for proteins which are slow to fold at the end of translation. This highlights the synergistic, yet distinct, roles of the ribosome and DnaK required to produce soluble, properly folded proteins which would otherwise be aggregation-prone on their own. Taken together, the work highlighted in this thesis sheds light on the unique mechanism by which the cell shapes, and ultimately traps, proteins into their native state during the earliest stages of protein life.
Scientists across disciplines have increasingly come to recognize the power of the protein. Current Protocols in Protein Science, a two-volume looseleaf manual, was developed in response to this revitalized interest and provides the most comprehensive collection of expert protein methods available. The publication covers both basic and advanced methods used in protein purification, characterization, and analysis as well as post-translational modification and structural analysis. More than 800 basic, support and alternate protocols have been carefully chosen for maximum applicability. Carefully edited, step-by-step protocols replete with material lists, expert commentaries, and safety and troubleshooting tips ensure that you can duplicate the experimental results in your own laboratory. Quarterly updates, which are filed into the looseleaf, keep the set current with the latest developments in protein science methods. The initial purchase includes one year of updates and then subscribers may renew their annual subscriptions. Current Protocols publishes a family of laboratory manuals for bioscientists, including Molecular Biology, Immunology, Human Genetics, Cytometry, Cell Biology, Neuroscience, Pharmacology, and Toxicology.
Book Synopsis Current Protocols in Protein Science Online by : Coligan
Download or read book Current Protocols in Protein Science Online written by Coligan and published by Current Protocols. This book was released on 2003-04-02 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Scientists across disciplines have increasingly come to recognize the power of the protein. Current Protocols in Protein Science, a two-volume looseleaf manual, was developed in response to this revitalized interest and provides the most comprehensive collection of expert protein methods available. The publication covers both basic and advanced methods used in protein purification, characterization, and analysis as well as post-translational modification and structural analysis. More than 800 basic, support and alternate protocols have been carefully chosen for maximum applicability. Carefully edited, step-by-step protocols replete with material lists, expert commentaries, and safety and troubleshooting tips ensure that you can duplicate the experimental results in your own laboratory. Quarterly updates, which are filed into the looseleaf, keep the set current with the latest developments in protein science methods. The initial purchase includes one year of updates and then subscribers may renew their annual subscriptions. Current Protocols publishes a family of laboratory manuals for bioscientists, including Molecular Biology, Immunology, Human Genetics, Cytometry, Cell Biology, Neuroscience, Pharmacology, and Toxicology.
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 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.