Design Structural Characterization And Application Of High Symmetry Protein Nanocages
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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.
Protein Cages
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Author : Takafumi Ueno
language : en
Publisher: Springer Nature
Release Date : 2023-06-12
Protein Cages written by Takafumi Ueno and has been published by Springer Nature this book supported file pdf, txt, epub, kindle and other format this book has been release on 2023-06-12 with Science categories.
This volume provides the latest methods for synthesis, structural analysis, and elucidation of the mechanism. Chapters guide readers through methods on protein cages for nanotechnology, analyze designed protein cages, determine the structures, and even perform theoretical analysis. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Protein Cages: Methods and Protocols aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.
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.
Protein Design
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Author : Valentin Köhler
language : en
Publisher: Humana
Release Date : 2016-09-17
Protein Design written by Valentin Köhler and has been published by Humana this book supported file pdf, txt, epub, kindle and other format this book has been release on 2016-09-17 with Science categories.
Protein Design: Method and Applications, Second Edition expands upon the previous edition with current, detailed ideas on how to approach a potential protein design project. With new chapters on metals as structure-forming elements and functional sites, the design and characterization of fluorinated proteins, top-down symmetric deconstruction and the design of protein libraries and novel or repurposed enzymes. Written in the highly successful Methods in Molecular Biologyseries format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Thorough and intuitive, Protein Design: Method and Applications, Second Edition provides a number of practical protocols and instructive reviews to aid in the creation of new experiments.
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.
Design And Functionalization Of Natural And Synthetic Protein Nanocages
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Author : Justin Evan Miller
language : en
Publisher:
Release Date : 2023
Design And Functionalization Of Natural And Synthetic Protein Nanocages written by Justin Evan Miller and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2023 with categories.
Biological containers known as protein cages are widespread in nature-displaying unique functionality via their ability to compartmentalize molecules in an internal lumen volume. Composed of repeated copies of a small number of protein subunits arranged symmetrically, protein cages are relatively simple in composition, and yet complex in structure. In nature these assemblies function in transport and protection, storage of important molecular cargo, and sequestration of metabolic reactions. These demonstrations of incredible functionality have inspired researchers seeking to engineer complex protein architectures with diverse functionality to direct their attention towards symmetric protein cages. Resulting from these efforts, a modest number of protein engineering studies have succeeded in replicating these structures de novo in hopes of applying them to the fields of medicine and biotechnology; however, developing applications for these assemblies that outperform existing technologies remains a challenging exercise. The primary aims of my thesis work are the design and development of applications for symmetric protein cages with a particular focus on mimicking natural functionalities not yet recreated via artificial protein design methods. We note the limitations of prevalent protein design methods, and technological advances that may revolutionize the field.The first chapter of this thesis is a brief introduction to protein cages as platforms for engineering, as well as efforts to design them de novo in the laboratory. In the second chapter of this thesis we sought to advance on existing methods for protein cage engineering by designing new cages with novel interface construction. These cages incorporate a grafted interface onto each of two components making up the cages. In this work we describe challenges associated with direct rigid linkage of protein components via alpha helical extension and discuss whether the technique is a viable strategy for future cage design efforts. Next we attempt a novel approach to addressing a long-standing challenge in the field of protein design: engineering structurally responsive protein cages. The natural world holds numerous examples of protein cages that can assemble and disassemble in response to specific stimuli, but recreating this effect in the laboratory has proven challenging. In chapter three we present work on the design of protein cages that disassemble in response to a specifically chosen target protease. This work related to protein cage disassembly has implications in therapeutic design, as proteases are common therapeutic targets, and developing systems responsive to diseases biomarkers could be of great utility. In the next chapter we expand our focus on cage disassembly to other types of triggers for cage disassembly. In this work, we devise a system utilizing antibody-mimetic molecules called DARPins that allows for the design of protein cages that disassemble in response to any protein of interest. The work in this chapter focuses on a natural cage assembly (sulfur oxygenase) as a platform for development of this effect. The fifth chapter of this thesis covers work related to designing protein-cage based nanoparticles to be used as immunosorbents for patients on dialysis. The work addresses a clinical need for methods to remove the protein beta-2 microglobulin from the blood, as high concentrations are common in patients on long-term hemodialysis therapy, but can be toxic. We describe a protein cage that displays nanobodies on its exterior surface to generate beta-2 microglobulin adsorbent cages (BACs). In conclusion, the research performed in this dissertation work seeks to advance on existing methods for protein cage engineering by designing new cages with novel interface construction. I then used previously described artificial protein cages to demonstrate triggerable protein cages with dynamic assembly properties that are responsive to molecular stimuli, as well as developing of an immunosorbent nanoparticle with clinical applications.
Protein Self Assembly
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Author : Jennifer J. McManus
language : en
Publisher: Humana
Release Date : 2020-08-08
Protein Self Assembly written by Jennifer J. McManus and has been published by Humana this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020-08-08 with Science categories.
This volume explores experimental and computational approaches to measuring the most widely studied protein assemblies, including condensed liquid phases, aggregates, and crystals. The chapters in this book are organized into three parts: Part One looks at the techniques used to measure protein-protein interactions and equilibrium protein phases in dilute and concentrated protein solutions; Part Two describes methods to measure kinetics of aggregation and to characterize the assembled state; and Part Three details several different computational approaches that are currently used to help researchers understand protein self-assembly. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Thorough and cutting-edge, Protein Self-Assembly: Methods and Protocols is a valuable resource for researchers who are interested in learning more about this developing field.
Encyclopedic Handbook Of Emulsion Technology
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Author : Johan Sjoblom
language : en
Publisher: CRC Press
Release Date : 2001-03-16
Encyclopedic Handbook Of Emulsion Technology written by Johan Sjoblom and has been published by CRC Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2001-03-16 with Science categories.
A discussion of fundamental characteristics, theories and applications for liquid-liquid colloidal dispersions. It profiles experimental and traditional measurement techniques in a variety of emulsified systems, including rheology, nuclear magnetic resonance, dielectric spectroscopy, microcalorimetry, video enhanced microscopy, and conductivity.
Structural Dna Nanotechnology
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Author : Nadrian C. Seeman
language : en
Publisher: Cambridge University Press
Release Date : 2015
Structural Dna Nanotechnology written by Nadrian C. Seeman and has been published by Cambridge University Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2015 with Computers categories.
Written by the founder of the field, this is a comprehensive and accessible introduction to structural DNA nanotechnology.
Macromolecular Protein Complexes
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Author : J. Robin Harris
language : en
Publisher: Springer
Release Date : 2017-03-07
Macromolecular Protein Complexes written by J. Robin Harris and has been published by Springer this book supported file pdf, txt, epub, kindle and other format this book has been release on 2017-03-07 with Science categories.
This volume of the established Subcellular Biochemistry series presents 20 chapters dealing with a broad range of interesting protein complexes. It will enable researchers to readily appreciate the major contribution from both X-ray crystallography and cryo-electron microscopy in this field of study. The biological significance of these structural studies is emphasised throughout the book. The diversity of the material included here indicates the breadth of this field and the tremendous progress that has been made in recent years. The book is directed primarily to advanced students and researchers in structural biology, and others in the biochemical sciences. It will be supplemented by other related books within the Subcellular Biochemistry series. One of the Editors (JM-W) is actively involved in structural biology and the other (JRH), as a retired academic and the Series Editor of Subcellular Biochemistry, has long experience at editing multi-author books./div