| 期刊全称 | Bacterial Amyloids | | 期刊简称 | Methods and Protocol | | 影响因子2023 | Véronique Arluison,Frank Wien,Andrés Marcoleta | | 视频video | http://file.papertrans.cn/181/180253/180253.mp4 | | 发行地址 | Includes cutting-edge techniques.Provides step-by-step detail essential for reproducible results.Contains key implementation advice from the experts | | 学科分类 | Methods in Molecular Biology | | 图书封面 |  | | 影响因子 | This detailed volume introduces different methods to assess the structure and folding of bacterial amyloids and analyze their functions, either in vitro or in vivo. Despite their initial association with neurodegenerative diseases, there is now increasing evidence of alternative roles for amyloids, with beneficial aspects for cells. In particular, prokaryotes use amyloids as functional assemblies, where they are key players in the cell‘s physiology. Written for the highly successful .Methods in Molecular Biology. series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. .Authoritative and up-to-date, .Bacterial Amyloids: Methods and Protocols. is an ideal guide for experts and novices alike to further explore these crucial protein assemblies.. | | Pindex | Book 2022 |
| 1 |
Front Matter |
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Abstract
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| 2 |
,Study of Amyloid Fibers Using Atomic Force Microscopy, |
Daniel G. Cava,Marisela Vélez |
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Abstract
Atomic force microscopy (AFM) provides high-resolution images of the topography of amyloid fibers adsorbed on surfaces. This information is very useful to study their molecular assembly under various conditions. This chapter describes the basic protocols required to deposit fibers on flat surfaces and discusses some of the practical issues required to operate a good commercial microscope setup to obtain appropriate high-resolution AFM topographic images of amyloid fibers.
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| 3 |
,Cryo-electron Microscopy to Analyze the Structure of Bacterial Amyloids In Vitro, |
Antoine Cossa,Sylvain Trépout |
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Abstract
Amyloid fibrils are aggregates of proteins or peptides. In humans, they are associated with various pathologies ranging from neurodegenerative diseases such as Alzheimer’s and Parkinson’s to systemic diseases like type 2 diabetes. In bacteria, amyloids can exert functional roles such as biofilm formation or gene regulation. Up to now, the aggregation mechanism leading to amyloid fibril formation is poorly understood as proteins with different amino acid sequences can fold into similar 3D structures. Understanding the formation of amyloid fibrils constitutes a central challenge for fighting major human health issues such as neurodegenerative diseases and biofilm formation in ports (implantable chambers). Since the dogma linking protein sequence, 3D structure, and function is increasingly disrupted by the growing understanding of the importance of disordered domains in proteins, it is crucial to possess a method capable of building accurate atomic models of amyloids. Aided by the leap forward of cryo-electron microscopy (cryo-EM), which can now routinely achieve sub-nanometric resolutions, it has become the method of choice for studying amyloids. In this chapter, we use the Hfq prote
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| 4 |
,Monitoring Molecular Assembly of Biofilms Using Quartz Crystal Microbalance with Dissipation (QCM-D |
Esra Yuca,Urartu Özgür Şafak Şeker |
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Abstract
The structure and the functionality of biofilm proteins, the main components of the extracellular matrix, can be tuned by protein engineering. The use of binding kinetics data has been demonstrated in the characterization of recombinantly produced biofilm proteins to control their behavior on certain surfaces or under certain conditions. Quartz crystal microbalance with dissipation monitoring (QCM-D) allows measuring the change in resonance frequency and the energy loss and distribution upon the interaction of molecules with the surface. The characterization of the molecular assembly of curli biofilm proteins on different surfaces using QCM-D is presented here as a detailed protocol. The experimental procedure detailed in this chapter can be applied and modified for other biofilm proteins or subunits to determine their surface adsorption and kinetic binding characteristics.
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| 5 |
,Measuring the Size and Spontaneous Fluctuations of Amyloid Aggregates with Fluorescence Correlation |
Vicky Vishvakarma,Sudipta Maiti |
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Abstract
Bacterial amyloids decorate the cell surface of many bacteria by forming functional amyloid fibers. These amyloids have structural and biochemical similarities with many disease-related amyloids in eukaryotes. Amyloid aggregation starts at the individual monomer level, and the end product is the amyloid fibril. The process of amyloid aggregation involves a continuous increase of the aggregate size, and therefore size is a critical parameter to measure in aggregation experiments. Also, our understanding of the aggregation process, and our ability to design interventions, can benefit from a measurement of the conformational dynamics of proteins undergoing aggregation. Fluorescence correlation spectroscopy (FCS) is perhaps the most sensitive and rapid technique available currently for this purpose. It can measure the average size and the size distribution of molecules and aggregates down to sub-nm length scales and can also measure fast nanosecond time-scale conformational dynamics, all in an equilibrium solution. FCS achieves this by measuring the fluorescence intensity fluctuations of freely diffusing protein molecules in an optically defined microscopic probe volume in a solution.
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| 6 |
,Determining the Stoichiometry of Amyloid Oligomers by Single-Molecule Photobleaching, |
Arpan Dey,Sudipta Maiti |
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Abstract
Small oligomers are the initial intermediates in the pathway to amyloid fibril formation. They have a distinct identity from the monomers as well as from the protofibrils and the fibrils, both in their structure and in their properties. In many cases, they play a crucial biological role. However, due to their transient nature, they are difficult to characterize. “Oligomer” is a diffuse definition, encompassing aggregates of many different sizes, and this lack of precise definition causes much confusion and disagreement between different research groups. Here, we define the small oligomers as “.”-mers with . < 10, which is the size range in which the amyloid proteins typically exist at the initial phase of the aggregation process. Since the oligomers dynamically interconvert into each other, a solution of aggregating amyloid proteins will contain a distribution of sizes. A precise characterization of an oligomeric solution will, therefore, require quantification of the relative population of each size. Size-based separation methods, such as size-exclusion chromatography, are typically used to characterize this distribution. However, if the interconversion between oligomers of differ
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| 7 |
,Structural Information on Bacterial Amyloid and Amyloid-DNA Complex Obtained by Small-Angle Neutron |
Tatsuhito Matsuo,Véronique Arluison,Frank Wien,Judith Peters |
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Abstract
Small-angle scattering is a powerful technique to obtain structural information on biomacromolecules in aqueous solution at the sub-nanometer and nanometer length scales. It provides the sizes and overall shapes of the scattering particles. While small-angle X-ray scattering (SAXS) has often been used for structural analysis of a single-component system, small-angle neutron scattering (SANS) has been used to reveal the internal organization of a multicomponent system such as protein-protein and protein-DNA complexes. This is due to the fact that the neutron scattering length is largely different between hydrogen and deuterium, and thus it allows to make a specific component in complexes “invisible” to neutrons by changing the H.O/D.O ratio in the solvent with or without molecular deuteration. In this chapter, we describe a method to characterize the biomolecular structures using SANS and SAXS, in particular, focusing on fibrillar proteins such as bacterial amyloids and their complexes with nucleic acids.
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| 8 |
,Fiber Diffraction and Small-Angle Scattering for Structural Investigation of Bacterial Amyloids, |
Tatsuhito Matsuo,Judith Peters |
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Abstract
X-ray/neutron fiber diffraction and small-angle X-ray/neutron scattering are widely used to investigate the molecular structure of fibrous proteins, including amyloid fibrils. However, there is sometimes confusion between these two techniques despite the fact that sample conditions and the content of the information obtained are not the same. In this brief chapter, we present the differences in sample conditions between these two methods, and their effects on experimentally obtained diffraction or scattering patterns, emphasizing the degree of disorder in the samples.
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| 9 |
,Supported Lipid Bilayers (SLBs) to Study Amyloid-Lipid Membrane Interactions with Atomic Force Micr |
Daniel G. Cava,Marisela Vélez |
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Abstract
Supported lipid bilayers (SLBs) are model membrane systems that can be used to study the interaction between amyloid fibers and membranes with atomic force microscopy (AFM). This chapter describes the preparation of SLBs on mica that can then be used as a substrate for fiber absorption. AFM can then be used to study the topography of the lipid-protein surface to study the evolution of the fibers, as well as the modifications on the membrane induced by their presence.
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| 10 |
,Characterization of Bacterial Amyloids by Nano-infrared Spectroscopy, |
Vincent Raussens,Jehan Waeytens |
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Abstract
Atomic force microscopy has been used for decades to study the topography of proteins during aggregation but with a lack of information on the secondary structure. On the contrary, infrared spectroscopy was able to study structural changes during the aggregation, but this analysis is complicated due to the presence of different species in mixtures and the poor spatial (~μm) resolution of the FTIR microscopy. Recently, Professor Alexandre Dazzi combined those techniques in the so-called AFM-IR. This method allows acquiring IR spectra at the nanometric scale and becomes a new standard method for the characterization of amyloid fibrils and, more generally, for the aggregation of proteins.
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| 11 |
,High-Throughput Screening of Heterologous Functional Amyloids Using , |
Elizabeth A. Yates,Luis A. Estrella,Christopher R. So |
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Abstract
remains one of the most widely used workhorse microorganisms for the expression of heterologous proteins. The large number of cloning vectors and mutant host strains available for . yields an impressively wide array of folded globular proteins in the laboratory. However, applying modern functional screening approaches to interrogate insoluble protein aggregates such as amyloids requires the use of nonstandard expression pathways. In this chapter, we detail the use of the curli export pathway in . to express a library of gene fragments and variants of a functional amyloid protein to screen sequence traits responsible for aggregation and the formation of nanoscale materials.
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| 12 |
,Evaluation of Amyloid Inhibitor Efficiency to Block Bacterial Survival, |
Florent Busi,Florian Turbant,Jehan Waeytens,Omar El Hamoui,Frank Wien,Véronique Arluison |
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Abstract
Amyloid inhibitors, such as the green tea compound epigallocatechin gallate EGCG, apomorphine or curlicide, have antibacterial properties. Conversely, antibiotics such as tetracycline derivatives or rifampicin also affect eukaryotic amyloids formation and may be used to treat neurodegenerative diseases. This opens the possibility for existing drugs to be repurposed in view of new therapy, targeting amyloid-like proteins from eukaryotes to prokaryotes and conversely. Here we present how to evaluate the effect of these amyloid-forming inhibitors on bacterial amyloid self-assemblies in vitro and on bacterial survival. The different approaches possible are presented.
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| 13 |
,Anti-Amyloid Drug Screening Methods Using Bacterial Inclusion Bodies, |
Ana B. Caballero,Patrick Gamez,Raimon Sabate,Alba Espargaró |
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Abstract
Amyloid aggregation is linked to a number of human disorders that range from non-neurological illnesses such as type 2 diabetes to neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. The formation of insoluble protein aggregates with amyloid conformation inside bacteria, namely, in bacterial inclusion bodies, offers the possibility to use bacteria as simple models to study amyloid aggregation processes and potential effects of both anti-amyloid drugs and/or pro-aggregative compounds. This chapter describes fast, simple, inexpensive, highly reproducible, and tunable in vitro and in cellulo methods that use bacterial inclusion bodies as preliminary screening tools for anti-amyloid drugs.
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| 14 |
,Determination of Effects and Mechanisms of Action of Bacterial Amyloids on Antibiotic Resistance, |
Krzysztof Kubiak,Lidia Gaffke,Karolina Pierzynowska,Zuzanna Cyske,Łukasz Grabowski,Katarzyna Kosznik |
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Abstract
Bacterial functional amyloids, apart from their many other functions, can influence the resistance of bacteria to antibiotics and other antibacterial agents. Mechanisms of modulation of susceptibility of bacterial cells to antimicrobials can be either indirect or direct. The former mechanisms are exemplified by the contribution of functional amyloids to biofilm formation, which may effectively prevent the penetration of various compounds into bacterial cells. The direct mechanisms include the effects of bacterial proteins revealing amyloid-like structures, like the C-terminal region of the . Hfq protein, on the expression of genes involved in antibiotic resistance. Therefore, in this paper, we describe methods by which effects and mechanisms of action of bacterial amyloids on antibiotic resistance can be studied. Assessment of formation of biofilms, determination of the efficiency of antibiotic resistance in solid and liquid media, and determination of the effects on gene expression at levels of mRNA abundance and stability and protein abundance are described.
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| 15 |
,Design and Testing of Synthetic Catalytic Amyloids Based on the Active Site of Enzymes, |
Claudio Castillo-Caceres,Eva Duran-Meza,Rodrigo Diaz-Espinoza |
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Abstract
The amyloid fold is nowadays recognized as an alternative conformation accessible to different proteins and peptides. The highly stable and ordered structural organization of amyloid fibrils can be exploited for the design of novel nanomaterials with emergent properties. Recent works have demonstrated that the functional features of the active site of enzymes can be partially recreated using this fold as a scaffold to develop catalytically active amyloids. We describe in this chapter a protocol to design functionally active amyloids that emerge from the self-assembly in vitro of synthetic peptides with sequences based on the active site of enzymes. Using this protocol, we show the development of amyloids that catalyze the metal-dependent hydrolysis of the phosphoanhydride bonds of nucleoside triphosphates.
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| 16 |
,Analysis of Bacterial Amyloid Interaction with Lipidic Membrane by Orientated Circular Dichroism an |
Jehan Waeytens,Florian Turbant,Véronique Arluison,Vincent Raussens,Frank Wien |
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Abstract
Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and orientated circular dichroism (OCD) are complementary spectroscopies widely used for the analysis of protein samples such as the amyloids commonly renowned as neurodegenerative agents. Determining the secondary structure content of proteins, such as aggregated β-sheets inside the amyloids and in various environments, including membranes and lipids, has made these techniques very valuable and complemental to high-resolution techniques such as nuclear magnetic resonance (NMR), X-ray crystallography, and cryo-electron microscopy. FTIR and CD are extremely sensitive to structural changes of proteins due to environmental changes. Furthermore, FTIR provides information on lipid modifications upon protein binding, whereas synchrotron radiation CD (SRCD) and OCD are sensitive to the subtle structural changes occurring in β-sheet-rich proteins and their orientation or alignment with lipid bilayers. FTIR and CD techniques allow the identification of parallel and antiparallel β-sheet content and are therefore complementary. In this chapter, we present FTIR and CD/OCD applications to study the interactions of bacteria
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| 17 |
,Direct, Rapid, and Simple Evaluation of the Expression and Conformation of Beta-Amyloid in Bacteria |
Christophe Sandt,David Partouche,Véronique Arluison |
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Abstract
The expression and conformation of bacterial proteins and peptides can be monitored in situ by Fourier transform infrared spectroscopy (FTIR), provided that the concentration of the protein of interest is sufficient. Here, we describe a simple protocol to analyze the conformation adopted by a specific amyloid protein in . cells, the pleiotropic regulator Hfq.. cells expressing Hfq under an inducible promoter are analyzed. The change in protein conformation is analyzed by comparing the different populations versus controls (i.e., Δ. cells, totally devoid of the Hfq protein) by difference spectroscopy, second derivation, curve-fitting, and principal component analysis. All the analyses were performed in the free, open-source software Quasar. We describe the detailed protocol for analyzing the data in Quasar. We show that the specific absorption of the β-amyloid conformation can be easily detected in the WT-Hfq, with bands at 1624 cm. and 1693 cm. indicating the presence of both parallel and antiparallel β-sheets. Furthermore, we show that FTIR spectroscopy is sensitive enough to probe the conformation of an amyloid protein backbone in vivo and to analyze its conformation in situ, dir
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| 18 |
,Assessment of Intracellular Amyloid Formation in Fixed and Live Bacteria Using Fluorescence Microsc |
Josefina Marín,Paulina Aguilera,Rosalba Lagos,Andrés Marcoleta |
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Abstract
Although amyloid aggregation has been generally associated with protein misfolding and neurodegenerative diseases in mammals, bacteria and other organisms have harnessed amyloidogenesis to perform diverse biological processes. These functional amyloids, some of them secreted and others intracellular, require that the producing cells keep aggregation under control in the cytoplasm upon protein translation, preventing their inherent toxicity. Thus, it is highly relevant to understand how intracellular amyloid formation occurs and is regulated, its metabolic consequences, and the formation dynamics and fate of the amyloid inclusions upon cell division. This chapter describes methods leveraging fluorescence microscopy and fixed- or live-cell imaging to monitor intracellular amyloid formation in bacterial cells.
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| 19 |
,Identification of Aggregation-Prone and Gatekeeper Residues in Bacterial Amyloids Using Site-Direct |
Paulina Aguilera,Josefina Marín,Rosalba Lagos,Andrés Marcoleta |
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Abstract
Bacterial functional amyloids are remarkable examples of how amyloid aggregation can be kept under control and even leveraged to perform diverse biological processes. In this context, it is highly relevant to understand how amyloidogenesis is modulated by relevant factors, including key amino acids promoting or preventing aggregation. This chapter describes a methodology to identify critical residues for amyloid formation in bacterial proteins, based on mutant construction guided by bioinformatics prediction, their expression in bacteria, and their analysis by flow cytometry. Additionally, we describe a simple downstream analysis of selected mutants to assess their in vitro aggregation properties upon protein purification. We applied the proposed methodology to identify critical residues modulating the aggregation of the antimicrobial peptide microcin E492, a well-studied model of bacterial amyloids.
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| 20 |
,Probing DNA-Amyloid Interaction and Gel Formation by Active Magnetic Wire Microrheology, |
Milad Radiom,Evdokia K. Oikonomou,Arnaud Grados,Mathieu Receveur,Jean-François Berret |
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Abstract
Recent studies have shown that bacterial nucleoid-associated proteins (NAPs) can bind to DNA and result in altered structural organization and bridging interactions. Under spontaneous self-assembly, NAPs may also form anisotropic amyloid fibers, whose effects are still more significant on DNA dynamics. To test this hypothesis, microrheology experiments on dispersions of DNA associated with the amyloid terminal domain (CTR) of the bacterial protein Hfq were performed using magnetic rotational spectroscopy (MRS). In this chapter, we survey this microrheology technique based on the remote actuation of magnetic wires embedded in a sample. MRS is interesting as it is easy to implement and does not require complex procedures regarding data treatment. Pertaining to the interaction between DNA and amyloid fibers, it is found that DNA and Hfq-CTR protein dispersions behave like a gel, an outcome that suggests the formation of a network of amyloid fibers cross-linked with the DNA strands. In contrast, the pristine DNA and Hfq-CTR dispersions behave as purely viscous liquids. To broaden the scope of the MRS technique, we include theoretical predictions for the rotation of magnetic wires regar
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