Design Characterization And Applications Of Symmetric Protein Scaffolds A
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Design Characterization And Applications Of Symmetric Protein Scaffolds A
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Author : David Leibly
language : en
Publisher:
Release Date : 2016
Design Characterization And Applications Of Symmetric Protein Scaffolds A written by David Leibly and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2016 with categories.
Proteins are essential macromolecules for all living organisms. They provide cellular structure and perform most of the metabolic functions essential for all life. The importance of proteins makes them the most studied and exploited macromolecules. We can exploit the structure of a protein to design a specific therapeutic to treat a disease or we can use proteins as biocatalysts for the efficient creation of molecules. In many instances, these applications of proteins are difficult to achieve. The work in this dissertation focuses on the development and evaluations of novel techniques to aid in the study and use of proteins. The first part of this dissertation focus on the creation of a series of symmetric oligomers to be used as crystallization scaffolds. Such scaffolds are intended to induce their symmetry onto asymmetric protein crystallization target proteins. The ability to determine the crystal structure can be essential for the creation of new targeted drugs or the better understanding of a biological process. Unfortunately many proteins fail to crystallize for reasons that are not well understood. It is thought that such induction of symmetry and variety of geometrically distinct scaffolds will aid in the crystallization of difficult-to-crystallize proteins. Preliminary results of these novel scaffolds and existing scaffolds are described. In the second part, applications of symmetric scaffolds for the creation of enzymatic materials are presented. These purely proteinaceous assemblies are designed to replicate previous described enzyme encapsulating materials. These materials typically improve enzyme reaction rates and product extraction. The final part of the dissertation focuses on the shell protein PduA from the 1,2-propanediol-utilization bacterial microcompartment (MCPs). These MCPs encapsulate metabolic pathways and contain volatile or toxic pathway intermediates. Research into turning these MCPs into bioreactors containing non-native enzymes is ongoing in many labs. Full realization of this technology relies on the encapsulation of new metabolic enzymes and transport of novel substrate and products through the shells. These processes are poorly understood, here structural studies of shell protein permutations. These permutations alter the topology of the shell protein allowing the scaffolding of proteins to the exterior surface of the MCPs. Finally, the efforts to elicited the interaction of specific targeting sequences to shell protein by x-ray crystallography are discussed.
Characterization Design And Application Of Natural And Engineered Symmetric Protein Complexes
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Author : Yuxi Liu
language : en
Publisher:
Release Date : 2018
Characterization Design And Application Of Natural And Engineered Symmetric Protein Complexes written by Yuxi Liu and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2018 with categories.
We frequently find proteins exist in oligomeric forms in nature. The abundance of dimers, trimers and tetramers with cyclic or dihedral symmetries in the Protein Data Bank is a good testimony. Even more, it is not rare to find proteins form highly ordered, symmetric, large complexes. These oligomeric forms are usually essential for their functions. Ferritin forms an octahedral cage with 24 subunits to store iron; some virus capsid proteins assemble into icosahedral cages; vaults, which are large dihedral particles widely conserved in eukaryotes, have biological functions yet to be discovered. These fascinating structures inspire three types of questions: How do individual subunits interact form such symmetric complexes? How can we reproduce such complexes with protein engineering? How do we put engineered symmetric protein complexes to application? My thesis work consists of projects addressing all three questions. My first project, described in Chapter 1, concerns bacterial microcompartments (MCP), which are large proteinaceous organelles enclosed by an icosahedral or pseudo-icosahedral shell. MCPs usually enclose special metabolic pathways that are inefficient or toxic in the cytosol. To do so, MCPs must form a sealed barrier with its shell proteins. It was hypothesized that at least one type of the proteins forming the shell of MCPs has to be pentameric instead of hexameric. Indeed, we proved that the BMV proteins, a family of protein highly conserved in MCP operons, formed pentamers in solution. Together with other crystallographic evidence, we conclude BMV proteins form pentamers to cap and seal the MCP shell. In addition to MCPs, I worked on another natural oligomeric protein, bactofilin. Bactofilins are fiber-forming proteins that are widely conserved among bacteria. These proteins have roles in diverse biological functions including but not limited to cell motility, cell wall synthesis and modification. Chapter 2 describe my preliminary biochemical and structural work on bactofilins. Next, I moved on to symmetry-based engineering protein complexes. In Chapter 3, I included a recent review paper on the theory and successes in symmetry-based protein engineering that I participated in preparing. Designed complexes need to be validated at high resolution with X-ray crystallography, but for a long time, the low yield and solubility of the designs complicated their validation. In Chapter 4, we show that mutating solvent-exposed side chains to charged amino acids improved the solubility of a previously low yield tetrahedral design and enabled validation by crystallography. Next, I advanced to a bigger challenge in designing symmetric nanoparticles--icosahedral particles. Icosahedral particles are made up of 60 asymmetric units, as compared to 12 in tetrahedral particles, making them much more difficult to design with accuracy. I was able to validate three different icosahedral design with crystallography, making them the largest designed protein assemblies ever crystallized to date. This work is described in Chapter 5. Additionally, I have made other independent design efforts, one to combine DNA and protein as building materials to design tetrahedral complexes, another to design protein sheets with layer group symmetry. These efforts are documented in Chapter 6.I In the last chapter, I utilized the validated tetrahedral designs as a scaffold in cryo-electron microscope (cryo-EM) for small targets. Despite recent advancements in cryo-EM techniques, small targets remain difficult. By arranging small targets around tetrahedral particles, we can overcome the size limit and provide multiple views to alleviate the commonly seen orientation preference. My project used a type of versatile adaptor protein, designed ankyrin repeat proteins (DARPins), to connect the tetrahedral particles to the imaging targets. We show that the resulting construct is amenable to structural analysis by single particle cryo-EM, allowing us to identify and solve the structure of the attached DARPin at near-atomic detail. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other proteins, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell. In conclusion, my thesis work contributes to the understanding of natural oligomeric complexes, expands our capacity in designing symmetric assemblies, and puts forward an example of a useful application of the designed assemblies.
Design Structural Characterization And Application Of High Symmetry Protein Nanocages
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Author : Kevin Alexander Cannon
language : en
Publisher:
Release Date : 2019
Design Structural Characterization And Application Of High Symmetry Protein Nanocages written by Kevin Alexander Cannon and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2019 with categories.
In nature, it is extremely common to find proteins that assemble into homo-oligomeric complexes from multiple copies of themselves. Almost half of known proteins form such complexes, most of which are cyclically or dihedrally symmetric. In some exceptional cases, however, protein molecules will self-assemble into much larger, closed three-dimensional geometries resembling the Platonic solids. Examples include icosahedral viral capsids, bacterial microcompartment shells, and octahedral ferritin assemblies. Protein scientists have studied and marveled at these exquisite protein cage structures for decades, and some have even ventured to produce novel types of protein cage assemblies unseen in nature through their own engineering efforts. In recent years, the field of protein design has seen striking progress in the development of design methodologies for taking proteins found in nature and modifying them to self-assemble into cages of tetrahedral, octahedral, or icosahedral point group symmetry, and these unique new types of protein assemblies are even beginning to find use in medicine, imaging, and biomaterials applications. My thesis work addresses both the design and application areas of the field of symmetric protein cage design. In Chapter 1, I include a recent review article on high symmetry protein assemblies, both natural and designed. A survey of all known structures in the Protein Data Bank that self-assemble into unique complexes with tetrahedral, octahedral, or icosahedral symmetry gives context for the types of biological functionality that seem to necessitate or benefit from such higher-order symmetries, although some intriguing mysteries remain unsolved. Our comparison of natural protein assemblies to the recent types of designed protein cages also emphasizes some unique properties of designed cages that remain unseen in natural assemblies. Next, I go on to describe some recent efforts to improve cage design methods to make cages that more reliably self-assemble into desired architectures when produced in the laboratory. In Chapter 2, we describe the design and characterization of two tetrahedral protein cage assemblies which were engineered to have hydrogen bonding networks at the interface between their two oligomeric components. These cages exhibit exceptionally high levels of soluble expression compared to most previous designed cages, but atomic structures solved by X-ray crystallography reveal some surprising deviations from the designed models. In Chapter 3, I describe efforts to design and characterize a protein icosahedron that self-assembles from 60 copies of a single designed protein subunit. To date, a designed icosahedral protein assembly formed from genetically fused protein oligomers (as opposed to multiple proteins self-assembling with a computationally designed interface) has yet to be validated in atomic detail. Challenges in achieving this goal have made it clear that novel, alternative design strategies are necessary. We describe the creation of a double-fusion protein containing dimer-, trimer-, and pentamer-forming protein domains in a single protein construct, which forms an icosahedral assembly when overexpressed in bacteria. The cage assembly is characterized by electron microscopy, small angle X-ray scattering, and other solution-state methods. I then go on to describe a project which applies a previously characterized tetrahedral protein cage scaffold as a platform for the multivalent display of cellulase enzymes. In Chapter 4, we describe the utilization of the T33-21 cage scaffold as a platform for covalently fusing other proteins to the exterior of the cage post-translationally using a sortase ligation method. In this work, we attach two different cellulase enzymes simultaneously to the cage scaffold and demonstrate increased synergy between the two enzymes in cellulose degradation assays. In conclusion, the work described in this thesis contributes to the ongoing development of novel design methodologies for engineering high symmetry protein cages, including some important lessons learned along the way, and goes on to describe the application of a designed cage scaffold as a multi-enzyme display platform for potential use in biofuels and other biomaterials technologies.
Theory Design And Characterization Of Protein Symmetry Combination Materials
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Author : Joshua Laniado
language : en
Publisher:
Release Date : 2020
Theory Design And Characterization Of Protein Symmetry Combination Materials written by Joshua Laniado and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020 with categories.
ABSTRACT OF THE DISSERTATIONTheory, Design and Characterization of Protein Symmetry Combination Materials by Joshua Laniado Doctor of Philosophy in the Molecular Biology Interdepartmental Doctoral Program: Biochemistry, Biophysics & Structural Biology University of California, Los Angeles, 2020 Professor Todd O. Yeates, Chair Nature has evolved a plethora of sophisticated protein complexes to carry out fundamental biological processes. While most of these exquisite macromolecular machines exhibit complex architectures, many are composed of only a few different types of subunits. Understanding how protein molecules combine to form these remarkable self-assembling structures only makes sense in the light of symmetry. By limiting the number of distinct interactions required between individual subunits, symmetry offers a simpler route for the evolution of supramolecular assemblies such as viral capsids and bacterial microcompartments. Principles of symmetry and self-assembly have invigorated recent efforts in molecular engineering giving rise to a growing suite of novel protein materials such as finite cages and extended crystalline arrays. These designed assemblies are rapidly finding applications in areas as diverse as vaccine design, atomic imaging, enzyme scaffolding and molecular delivery. Despite significant advances in computational approaches and design strategies, constructing these materials remains extremely challenging. Here, we address key experimental and theoretical limitations to improve the prospects for the routine design of novel symmetric protein materials. In Chapter 1, we review current methodologies for designing self-assembling protein nanomaterials. A first approach presented the idea that when two separate symmetric oligomers associate in some geometrically defined way, a structure with higher symmetry can be obtained through self-assembly. There, an alpha-helical linker is used to connect two oligomeric components and to control their relative geometry. A second approach does not involve genetic fusion but relies instead on the computational design of a novel protein-protein interface. After reviewing the successful constructions resulting from both methods, challenges and limitations are discussed. In the fusion approach, the inherent flexibility of the alpha helical linker can lead to the formation of unintended assemblies. Alternatively, the interface design strategy exhibits limited success in predicting viable protein interfaces. The prevalence of such limitations dramatically hinders the creation of novel materials, motivating the development of alternate strategies. In the next chapter, we introduce a new approach for the design of symmetric self-assembling nanomaterials. Building upon the fusion approach, the original alpha-helical linker is replaced with a heterodimeric coiled coil as an attempt to reduce flexibility. Further, the use of a known heterodimeric interface to combine component oligomers alleviates the challenges associated with de novo interface design. Ten symmetric protein cages were designed using this method among which two were structurally characterized. One design assembled as intended while the other crystallized in an alternate form. Geometric distinctions between the two help explain the different degrees of success, leading to crucial lessons and establishing clearer principles for the creation of novel nanoscale protein architectures. While some experimental aspects have been addressed, only a small fraction of the possible design space has been explored. That space, which is anticipated to offer a multitude of symmetry-based combinations, has not been described in theory. In Chapter 3, we articulate all of the possible kinds of protein-based materials that can be created by combining two symmetric oligomers. Specifically, 13 types of cages, 35 types of 2-D layers and 76 types of 3-D crystals are identified as possible targets for design. We lay out a complete rule set for constructing all such symmetry combination materials (SCMs) and introduce a unified system for parameterizing and searching the construction space for each case. This theoretical and computational study provides a blueprint for a blossoming area of macromolecular design. Owing to the complexity and our limited understanding of the rules that govern protein behavior, designing protein-protein interfaces remains challenging. Current approaches rely on empirical or knowledge-based energy functions and optimization algorithms that often fail to produce stable interfaces. On the other hand, there is growing evidence that the database of known protein structures is now sufficiently large to cover the structural landscape of protein interfaces. In Chapter 4, we argue that carefully-selected structural motifs can be used as templates for interface design. We introduce Nanohedra, a fragment-based docking tool that harnesses the power of our theoretical framework to enable the design of all possible SCMs. Prospective designs of symmetric materials are proposed along with a retrospective analysis of recent design studies. In this analysis, our tool recapitulates all successful designs while poorly ranking failed ones. With a user-friendly interface and a unified protocol for symmetric protein design, Nanohedra enables the creation of a universe of novel nanomaterials and opens new avenues for nanobiotechnology.
Characterisation And Design Of Tissue Scaffolds
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Author : Paul Tomlins
language : en
Publisher: Elsevier
Release Date : 2015-10-30
Characterisation And Design Of Tissue Scaffolds written by Paul Tomlins and has been published by Elsevier this book supported file pdf, txt, epub, kindle and other format this book has been release on 2015-10-30 with Technology & Engineering categories.
Characterisation and Design of Tissue Scaffolds offers scientists a useful guide on the characterization of tissue scaffolds, detailing what needs to be measured and why, how such measurements can be made, and addressing industrially important issues. Part one provides readers with information on the fundamental considerations in the characterization of tissue scaffolds, while other sections detail how to prepare tissue scaffolds, discuss techniques in characterization, and present practical considerations for manufacturers. Summarizes concepts and current practice in the characterization and design of tissue scaffolds Discusses design and preparation of scaffolds Details how to prepare tissue scaffolds, discusses techniques in characterization, and presents practical considerations for manufacturers
Software Algorithms For Design Of Symmetric Protein Complexes Applied To Cryo Electron Microscopy Scaffolds And Antibody Nanoparticles
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Author : Ivan Vulovic
language : en
Publisher:
Release Date : 2020
Software Algorithms For Design Of Symmetric Protein Complexes Applied To Cryo Electron Microscopy Scaffolds And Antibody Nanoparticles written by Ivan Vulovic and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020 with Genomics categories.
Symmetric Ligand Binding Using Tunable De Novo Designed Symmetric Protein Dimers
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Author : Derrick R. Hicks
language : en
Publisher:
Release Date : 2020
Symmetric Ligand Binding Using Tunable De Novo Designed Symmetric Protein Dimers written by Derrick R. Hicks and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020 with categories.
Cyclic two-fold (C2) symmetric ligands are common in nature, synthetic chemistry, and medicine. Additionally, we can now design millions of C2 symmetric peptides with an incredible diversity of sizes, shapes, and chemistries. De novo proteins capable of binding these C2 symmetric ligands could be useful in various applications, but scaffolds and methods to do this have been lacking. To solve this problem, I created a diverse set of C2 symmetric proteins with central cavities. I first designed curved repeat protein monomers sampling a continuum of curvatures, and then docked these into homodimers, generating a very wide range of C2 cavity shapes and sizes for functionalization. In total, 77 scaffolds were experimentally characterized, and of these, 23 (30%) appear to be folded as designed based on Small Angle X-ray Scattering data. Furthermore, crystallographic data for 4 designs (2 scaffolds and 2 functionalized binders) confirms the proteins fold as expected. A third scaffold design was determined to be monomeric by crystallographic analysis. Despite its failure to form the designed homodimer, the solved monomer was in close agreement with the design model. We believe that these diverse scaffolds provide a rich set of starting points for binding a very wide range of C2 compounds. Advantages of this conception are that the cavities can be very diverse in size, shape, and available sidechain chemistry, and as the protein hydrophobic core is separated from the pocket because the cavity lining residues are on the exterior of the monomers, functionalization to create binding interactions for specific compounds is unlikely to destabilize either the monomers or the dimer interface. Finally, we used these scaffolds to bind symmetric chlorophyll dimers, which could have applications in synthetic light-harvesting, as well as to bind a C2 symmetric peptide, which could become a platform for the creation of entirely bioorthogonal chemically induced dimers.
Symmetric Protein Assembly
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Author : Jennifer Erin Padilla
language : en
Publisher:
Release Date : 2003
Symmetric Protein Assembly written by Jennifer Erin Padilla and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2003 with categories.
Ionic Interactions In Natural And Synthetic Macromolecules
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Author : Alberto Ciferri
language : en
Publisher: John Wiley & Sons
Release Date : 2012-01-04
Ionic Interactions In Natural And Synthetic Macromolecules written by Alberto Ciferri and has been published by John Wiley & Sons this book supported file pdf, txt, epub, kindle and other format this book has been release on 2012-01-04 with Science categories.
This book is a comprehensive study of the subject of ionic interactions in macromolecules. The first parts of the book review and analyze the conventional treatments of fixed charges (e.g. in polyelectrolytes and polyampholytes), including screening and condensation by mobile ions. The interaction of ions with less polar sites on the macromolecule (e.g. amide bonds), and the origin of the lyotropic effects (focusing on binding versus condensation) will also be extensively addressed. The book also explores complex micellar organizations involving charged macromolecules (e.g. DNA) and low-molecular-weight ampholytes and strong protein associations. The resulting structures are relevant to a variety of functional biological systems and synthetic analogs. The contribution of electrostatic and hydrophobic interaction to the stability of proteins and other supramolecular structures will also be analyzed. There are chapters on applications such as deionization and cosmetic formulation. This 21-chapter book is divided into three sections: Fundamentals Mixed Interactions Functions and Applications
29th Annual Conference Of The German Crystallographic Society March 15 18 2021 Hamburg Germany
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Author : Deutsches Elektronen-Synchrotron-DESY
language : en
Publisher: Walter de Gruyter GmbH & Co KG
Release Date : 2021-04-19
29th Annual Conference Of The German Crystallographic Society March 15 18 2021 Hamburg Germany written by Deutsches Elektronen-Synchrotron-DESY and has been published by Walter de Gruyter GmbH & Co KG this book supported file pdf, txt, epub, kindle and other format this book has been release on 2021-04-19 with Science categories.
Zeitschrift für Kristallographie. Supplement Volume 41 presents the complete Abstracts of all contributions to the 29th Annual Conference of the German Crystallographic Society in Hamburg (Germany) 2021: - Plenary Talks - Microsymposia - Poster Session Supplement Series of Zeitschrift für Kristallographie publishes Abstracts of international conferences on the interdisciplinary field of crystallography.