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Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Useful structural information about the conformation of nucleic acids can be quickly acquired by circular and linear dichroism (CD/LD) spectroscopy. These techniques, rely on the differential absorption of polarised light and are indeed extremely sensitive to subtle changes in the structure of chiral biomolecules. Many CD analyses of DNA or DNA:protein complexes have been conducted with substantial data acquisitions. Conversely, CD RNA analysis are still scarce, despite the fact that RNA plays a wide cellular function. This chapter seeks to introduce the reader to the use of circular, linear dichroism and in particular the use of Synchrotron Radiation for such samples. The use of these techniques on small noncoding RNA (sRNA) will be exemplified by analyzing changes in base stacking and/or helical parameters for the understanding of sRNA structure and function, especially by translating the dynamics of RNA:RNA annealing but also to access RNA stability or RNA:RNA alignment. The effect of RNA remodeling proteins will also be addressed. These analyses are especially useful to decipher the mechanisms by which sRNA will adopt the proper conformation thanks to the action of proteins such as Hfq or ProQ in the regulation of the expression of their target mRNAs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 bacterial amyloids with membranes and lipids. Moreover, we show how to decipher the spectroscopic signals to obtain information on the molecular structure of amyloids and their interaction with lipids, addressing potential amyloid insertion into membranes and the lipid bilayer adjustments observed., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanofluidics is an emerging methodology to investigate single biomacromolecules without functionalization and/or attachment of the molecules to a substrate. In conjunction with fluorescence microscopy, it can be used to investigate structural and dynamical aspects of amyloid-DNA interaction. Here, we summarize the methodology for fabricating lab-on-chip devices in relatively cheap polymer resins and featuring quasi one-dimensional nanochannels with a cross-sectional diameter of tens to a few hundred nanometers. Site-specific staining of amyloid-forming protein Hfq with a fluorescence dye is also described. The methodology is illustrated with two application studies. The first study involves assembling bacterial amyloid proteins such as Hfq on double-stranded DNA and monitoring the folding and compaction of DNA in a condensed state. The second study is about the concerted motion of Hfq on DNA and how this is related to DNA's internal motion. Explicit details of procedures and workflows are given throughout., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 2 O/D 2 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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 Escherichia coli cells, the pleiotropic regulator Hfq.E. coli 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., Δhfq 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 -1 and 1693 cm -1 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, directly in bacterial cells, without the need for protein purification., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacterial chromosomal DNA is packed within a non-membranous structure, the nucleoid, thanks to nucleoid associated proteins (NAPs). The role of bacterial amyloid has recently emerged among these NAPs, particularly with the nucleoid-associated protein Hfq that plays a direct role in DNA compaction. In this chapter, we present a 3D imaging technique, cryo-soft X-ray tomography (cryo-SXT) to obtain a detailed 3D visualization of subcellular bacterial structures, especially the nucleoid. Cryo-SXT imaging of native unlabeled cells enables observation of the nucleoid in 3D with a high resolution, allowing to evidence in vivo the role of amyloids on DNA compaction. The precise experimental methods to obtain 3D tomograms will be presented., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies are fast techniques providing important information about the conformation of nucleic acids and proteins. These vibrational and electronic absorption spectroscopies are extremely sensitive to any change in molecular structure. While numerous reviews describe how to analyze DNA structure alone or in the presence of proteins using FTIR and CD, analyses of RNA are scarce. Nevertheless, RNA remodeling proteins are important factors involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) and FTIR to analyze how proteins may change RNA structure. These include the analysis of RNA melting, or stabilization, of change in helical parameters and base stacking. The effects on the structure of RNA remodeling proteins are also presented.

Fourier transform infrared (FTIR) spectroscopy has been widely used for the analysis of both protein and nucleic acid secondary structure. This is one of the vibration spectroscopy methods that are extremely sensitive to any change in molecular structure. While numerous reports describe how to proceed to analyze protein and deoxyribonucleic acid (DNA) structures using FTIR, reports related to the analyses of ribonucleic acids (RNAs) are few. Nevertheless, RNAs are versatile molecules involved in a multitude of roles in the cell. In this chapter, we present applications of FTIR for the structural analysis of RNA, including the analysis of helical parameters and noncanonical base pairing, often found in RNA. The effect of temperature pretreatment, which has a great impact on RNA folding, will also be discussed.

Circular dichroism (CD) spectroscopy is a fast and simple technique providing important information about the conformation of nucleic acids, proteins, sugars, lipids, and their interactions between each other. This electronic absorption spectroscopy method is extremely sensitive to any change in molecular structure containing asymmetric molecules. While numerous reviews describe how to analyze deoxyribonucleic acid (DNA) structures using CD, analyses of ribonucleic acids (RNAs) are scarce. Nevertheless, RNAs are important molecules involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) extending the spectral range down to 170 nm, improving structural analysis of RNA, including the analysis of helical parameters and alternative structures found in RNA. The effects of temperature to measure thermodynamic parameters and analyze ribonucleoprotein complexes will also be presented.

Atomic force and transmission electron microscopies (AFM/TEM) are powerful tools to analyze RNA-based nanostructures. While cryo-TEM analysis allows the determination of near-atomic resolution structures of large RNA complexes, this chapter intends to present how RNA nanostructures can be analyzed at room temperature on surfaces. Indeed, TEM and AFM analyses permit the conformation of a large population of individual molecular structures to be observed, providing a statistical basis for the variability of these nanostructures within the population. Nevertheless, if double-stranded DNA molecular imaging has been described extensively, only a few investigations of single-stranded DNA and RNA filaments have been conducted so far. Indeed, technique for spreading and adsorption of ss-molecules on AFM surfaces or TEM grids is a crucial step to avoid disturbing RNA conformation on the surface. In this chapter, we present a specific method to analyze RNA assemblies and RNA-protein complexes for molecular microscopies.

Post-transcriptional control of gene expression by small regulatory noncoding RNA (sRNA) needs protein accomplices to occur. Past research mainly focused on the RNA chaperone Hfq as cofactor. Nevertheless, recent studies indicated that other proteins might be involved in sRNA-based regulations. As some of these proteins have been shown to self-assemble, we describe in this chapter protocols to analyze the nano-assemblies formed. Precisely, we focus our analysis on Escherichia coli Hfq as a model, but the protocols presented here can be applied to analyze any polymer of proteins. This chapter thus provides a guideline to develop commonly used approaches to detect prokaryotic protein self-assembly, with a special focus on the detection of amyloidogenic polymers.

In recent years, single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful technique to study macromolecular interactions. The chief advantages of smFRET analysis compared to bulk measurements include the possibility to detect sample heterogeneities within a large population of molecules and the facility to measure kinetics without needing the synchronization of intermediate states. As such, the methodology is particularly well adapted to observe and analyze RNA/RNA and RNA/protein interactions involved in small noncoding RNA-mediated gene regulation networks. In this chapter, we describe and discuss protocols that can be used to measure the dynamics of these interactions, with a particular emphasis on the advantages-and experimental pitfalls-of using the smFRET methodology to study sRNA-based biological systems.

Regulation of RNA turnover is of utmost importance for controlling the concentration of transcripts and consequently cellular protein levels. Among the processes controlling RNA decay, small noncoding regulatory RNAs (sRNAs) have recently emerged as major new players. In this chapter, we describe and discuss protocols that can be used to measure sRNA concentration in vivo and to assess sRNA decay rates in Gram-negative bacteria. Precisely, we focus our analyses on the Escherichia coli Gram-negative bacterium as a model. The information described in this chapter provides a guideline to help develop a protocol in order to assess these important parameters and to identify RNA-processing enzymes involved in sRNA degradation processes.

RNAs are flexible molecules involved in a multitude of roles in the cell. Specifically, noncoding RNAs (i.e., RNAs that do not encode a protein) have important functions in the regulation of biological processes such as RNA decay, translation, or protein translocation. In bacteria, most of those noncoding RNAs have been shown to be critical for posttranscriptional control through their binding to the untranslated regions of target mRNAs. Recent evidence shows that some of these noncoding RNAs have the propensity to self-assemble in prokaryotes. Although the function of this self-assembly is not known and may vary from one RNA to another, it offers new insights into riboregulation pathways. We present here the various approaches that can be used for the detection and analysis of bacterial small noncoding RNA self-assemblies.

The ability to study the localization and organization of proteins within the tiny cells of bacteria, such as Escherichia coli, has paved the way for a new and exciting era of prokaryotic cellular biology. Previously unrecognized levels of spatiotemporal and supramolecular organization of proteins have been revealed within the prokaryotic cell that had long been assumed as a "bag of enzymes." Immunofluorescence (IF) microscopy, which involves cellular immunostaining of native proteins with fluorescently labeled antibodies, is relatively laborious and requires cell fixation and highly specific antibodies. However, IF microscopy allows localization studies of native proteins expressed to their normal cellular levels, as opposed to labeling proteins with large fluorescent tag that can alter protein abundance dependent on changes in mRNAs and/or proteins stability, or whose detection can require overexpression of labeled proteins. In addition, when antibodies against native proteins are not available or lack specificity, epitope tags such as hemagglutinin (HA) or Flag can be used to label chromosomally expressed proteins. The short Flag- and HA-tag, eight or nine amino acids, are unlikely to interfere with the localization or function of the proteins. We describe and discuss here the use of fluorescence microscopy for determination of cellular organization of protein components of the E. coli RNA processing and degradation machinery. We present examples of cellular organization patterns visualized by light microscopy, either by IF microscopy of native and epitope-tagged proteins in fixed cells, or by fluorescence labeling of the proteins in live cells.