The Effects Of Macromolecular Crowding And System Volume On Protein Aggregation

Proteins in living cells perform an enormous variety of processes, among these is protein self-assembly or aggregation. While many proteins self-assemble as part of healthy biological function, malfunctioning proteins which pathologically self-assemble have been linked to numerous neurodegenerative diseases like Huntington's, Parkinson's, and Alzheimer's. We investigate the role of macromolecular crowders and system volume in the process of protein self-assembly. We present three major studies in this thesis: two on the role of macromolecular crowders in the self-assembly of actin and amyloid proteins using differential rate equations, and one utilizing a stochastic approach to study the effects of system volume and particle number on protein aggregation, as well as how crowders can change the microscopic details of reaction dynamics. We develop a model for the inclusion of crowding effects in kinetic rate constants and show remarkable fits to existing data for actin and nine amyloid-forming proteins in the presence of macromolecular crowders. We also show how fluctuations and overall dynamics change with system size and in the presence of crowders, and develop a stochastic method for determining the prevalence of individual reaction mechanisms and how they evolve over time. This reaction frequency method provides unique insight into the dynamics of the system and can be used to explain why certain aggregating systems behave as they do, and why their behavior changes in various conditions. Stochastic approaches, such as Gillespie's algorithm, may be necessary for studying the behavior of proteins in cells, as volumes of micelles and cellular compartments can be as small as 1pL, thus fluctuations in particle number become relevant and continuous rate approaches become less accurate. We also present a tool for user-friendly simulation and visualization of stochastic models of protein aggregation.

This book reviews current research on the important processes involved in neurodegenerative diseases (e.g. Alzheimer's disease) and the peptides and proteins involved in the amyloidogenic processes. It covers the design and developments of anti-amyloid inhibitors, and gives readers a fundamental understanding of the underlying oligomerization and aggregation processes of these diseases from both computational and experimental points 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.

Macromolecules can occupy a large fraction of the volume of a cell and this crowded environment influences the behavior and properties of the proteins, such as mechanical unfolding forces, thermal stability and rates of folding and diffusion. Although much is already known about molecular crowding, it is not well understood how it affects a protein's resistance to mechanical stress in a crowded environment and how the size of the crowders affect those changes. An atomic force microscope-based single molecule method was used to measure the effects of the crowding on the mechanical stability of a model protein, in this case I-27. As proteins tend to aggregate, single molecule methods provided a way to prevent aggregation because of the very low concentration of proteins in the solution under study. Dextran was used as the crowding agent with three different molecular weights 6kDa, 10 kDa and 40 kDa, with concentrations varying from zero to 300 grams per liter in a pH neutral buffer solution at room temperature. Results showed that the forces required to unfold biomolecules were increased when a high concentration of crowder molecules were added to the buffer solution and that the maximum force required to unfold a domain was when the crowder size was 10 kDa, which is comparable to the protein size. Unfolding rates obtained from Monte Carlo simulations showed that they were also affected in the presence of crowders. As a consequence, the energy barrier was also affected. These effects were most notable when the size of the crowder was 10 kDa, comparable to the size of the protein. On the other hand, distances to the transition state did not seem to change when crowders were added to the solution. The effect of Dextran on the energy barrier was modeled by using established theories such as Ogston's and scaled particle theory, neither of which was completely convincing at describing the results. It can be hypothesized that the composition of Dextran plays a role in the deviation of the predicted behavior with respect to the experimental data.

The cell can be viewed as a 'collection of protein machines' and understanding these molecular machines requires sophisticated cooperation between cell biologists, geneticists, enzymologists, crystallographers, chemists and physicists. To observe these machines in action, researchers have developed entirely new methodologies for the detection and the nanomanipulation of single molecules. This book, written by expert scientists in the field, analyses how these diverse fields of research interact on a specific example - RNA polymerase. The book concentrates on RNA polymerases because they play a central role among all the other machines operating in the cell and are the target of a wide range of regulatory mechanisms. They have also been the subject of spectacular advances in their structural understanding in recent years, as testified by the attribution of the Nobel prize in chemistry in 2006 to Roger Kornberg. The book focuses on two aspects of the transcription cycle that have been more intensively studied thanks to this increased scientific cooperation - the recognition of the promoter by the enzyme, and the achievement of consecutive translocation steps during elongation of the RNA product. Each of these two topics is introduced by an overview, and is then presented by worldwide experts in the field, taking the viewpoint of their speciality. The overview chapters focus on the mechanism-structure interface and the structure-machine interface while the individual chapters within each section concentrate more specifically on particular processes-kinetic analysis, single-molecule spectroscopy, and termination of transcription, amongst others. Specific attention has been paid to the newcomers in the field, with careful descriptions of new emerging techniques and the constitution of an atlas of three-dimensional pictures of the enzymes involved. For more than thirty years, the study of RNA polymerases has benefited from intense cooperation between the scientific partners involved in the various fields listed above. It is hoped that a collection of essays from outstanding scientists on this subject will catalyse the convergence of scientific efforts in this field, as well as contribute to better teaching at advanced levels in Universities.

Completely revised and expanded to reflect the latest advancements in the field, Polysaccharides: Structural Diversity and Functional Versatility, Second Edition outlines fundamental concepts in the structure, function, chemistry, and stability of polysaccharides and reveals new analytical techniques and applications currently impacting the cosmeti

This book makes a novel synthesis of the molecular aspects of the stress response and long term adaptation processes with the system biology approach of biological networks. Authored by an exciting mixture of top experts and young rising stars, it provides a comprehensive summary of the field and identifies future trends.

The first of its kind, this volume presents the latest research findings on the chaperonins, the best studied family of a class of proteins known as molecular chaperones. These findings are changing our view of some fundamental cellular processes involving proteins, especially how proteins fold into their functional conformations. Origins of the new view of protein folding Prokaryotic chaperonins Eukaryotic chaperonins Evolution of the chaperonins Refolding of denatured proteins Organelle biosynthesis Biomedical aspects

The topics covered by this volume include: protein destabilization at low temperatures; engineering the stability and function of Gene V Protein; free energy balance in protein folding; modelling protein stability as a heteropolymer collapse; stability of alpha helices; protein stability with T4 Lysozyme.