Small-angle X-ray scattering of engineered antigen-binding fragments: the case of glycosylated Fab from the Mannitou IgM antibody.

Monoclonal antibodies recognizing nonprotein antigens remain largely underrepresented in our understanding of the molecular repertoire of innate and adaptive immunity. One such antibody is Mannitou, a murine IgM that recognizes paucimannosidic glycans. In this work, we report the production and purification of the recombinant antigen-binding fragment (Fab) of Mannitou IgM (Mannitou Fab) and employ a combination of biochemical and biophysical approaches to obtain its initial structural characterization.

Effect of base methylation on binding and mobility of bacterial protein Hfq on double-stranded DNA.

Regulation of protein mobility is a fundamental aspect of cellular processes. In this study, we examined the impact of DNA methylation on the diffusion of nucleoid associated protein Hfq. This protein is one of the most abundant proteins that shapes the bacterial chromosome and is involved in several aspects of nucleic acid metabolism.

Use of Synchrotron Radiation Circular Dichroism to Analyze the Interaction and Insertion of Proteins into Bacterial Outer Membrane Vesicles.

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).

Angle-Resolved Linear Dichroism to Probe the Organization of Highly Ordered Collagen Biomaterials.

Controlling the assembly of high-order structures is central to soft-matter and biomaterial engineering. Angle-resolved linear dichroism can probe the ordering of chiral collagen molecules in the dense state. Collagen triple helices were aligned by solvent evaporation.

The role of membrane physiology in sHSP Lo18-lipid interaction and lipochaperone activity.

To cope with environmental stresses, organisms, including lactic acid bacteria such as O. oeni, produce stress proteins called HSPs. In wine, O.

A comprehensive investigation of the interactions of human serum albumin with polymeric and hybrid nanoparticles.

Nanoparticles (NPs) engineered as drug delivery systems continue to make breakthroughs as they offer numerous advantages over free therapeutics. However, the poor understanding of the interplay between the NPs and biomolecules, especially blood proteins, obstructs NP translation to clinics. Nano-bio interactions determine the NPs' in vivo fate, efficacy and immunotoxicity, potentially altering protein function.

The Amyloid Assembly of the Bacterial Hfq Is Lipid-Driven and Lipid-Specific.

Under specific conditions, some proteins can self-assemble into fibrillar structures called amyloids. Initially, these proteins were associated with neurodegenerative diseases in eucaryotes. Nevertheless, they have now been identified in the three domains of life.

Role of the Bacterial Amyloid-like Hfq in Fluoroquinolone Fluxes.

Due to their two-cell membranes, Gram-negative bacteria are particularly resistant to antibiotics. Recent investigations aimed at exploring new target proteins involved in Gram-negative bacteria adaptation helped to identify environmental changes encountered during infection. One of the most promising approaches in finding novel targets for antibacterial drugs consists of blocking noncoding RNA-based regulation using the protein cofactor, Hfq.

Circular and Linear Dichroism for the Analysis of Small Noncoding RNA Properties.

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.

The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation.

Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation.

Significant influence of four highly conserved amino-acids in lipochaperon-active sHsps on the structure and functions of the Lo18 protein.

To cope with environmental stresses, bacteria have developed different strategies, including the production of small heat shock proteins (sHSP). All sHSPs are described for their role as molecular chaperones. Some of them, like the Lo18 protein synthesized by Oenococcus oeni, also have the particularity of acting as a lipochaperon to maintain membrane fluidity in its optimal state following cellular stresses.

Hfq C-terminal region forms a β-rich amyloid-like motif without perturbing the N-terminal Sm-like structure.

Hfq is a pleitropic actor that serves as stress response and virulence factor in the bacterial cell. To execute its multiple functions, Hfq assembles into symmetric torus-shaped hexamers. Extending outward from the hexameric core, Hfq presents a C-terminal region, described as intrinsically disordered in solution.

Amyloid-like Hfq interaction with single-stranded DNA: involvement in recombination and replication in .

Interactions between proteins and single-stranded DNA (ssDNA) are crucial for many fundamental biological processes, including DNA replication and genetic recombination. Thus, understanding detailed mechanisms of these interactions is necessary to uncover regulatory rules occurring in all living cells. The RNA-binding Hfq is a pleiotropic bacterial regulator that mediates many aspects of nucleic acid metabolism.

Interactions and Insertion of Hfq into Outer Membrane Vesicles as Revealed by Infrared and Orientated Circular Dichroism Spectroscopies.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs.

Synchrotron radiation circular dichroism spectroscopy of oligonucleotides at millimolar concentrations.

Circular dichroism spectroscopy of nucleic acids has been traditionally performed at sample concentrations orders of magnitude lower than what occur in biological systems. While recent work from us demonstrated the flexibility of an adjustable sample cell that allowed for successful recording of CD spectra of an 18- and a 21-mer double stranded DNA sequences at around 1 mM, sample concentrations beyond 1 mM present a challenge for standard benchtop CD spectrometers. In the present work, the synchrotron radiation circular dichroism (SRCD) spectra were recorded for d(CG) and a mixed 18-mer double stranded DNA at 1, 5, and 10 mM in 100 mM or 4 M NaCl.

New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

Poldip2 was shown to be involved in oxidative signaling to ensure certain biological functions. It was proposed that, in VSMC, by interaction with the Nox4-associated membrane protein p22, Poldip2 stimulates the level of reactive oxygen species (ROS) production. In vitro, with fractionated membranes from HEK393 cells over-expressing Nox4, we confirmed the up-regulation of NADPH oxidase 4 activity by the recombinant and purified Poldip2.

Cryo soft X-ray tomography to explore Escherichia coli nucleoid remodeling by Hfq master regulator.

The bacterial chromosomic DNA is packed within a membrane-less structure, the nucleoid, due to the association of DNA with proteins called Nucleoid Associated Proteins (NAPs). Among these NAPs, Hfq is one of the most intriguing as it plays both direct and indirect roles on DNA structure. Indeed, Hfq is best known to mediate post-transcriptional regulation by using small noncoding RNA (sRNA).

NACDDB: Nucleic Acid Circular Dichroism Database.

The Nucleic Acid Circular Dichroism Database (NACDDB) is a public repository that archives and freely distributes circular dichroism (CD) and synchrotron radiation CD (SRCD) spectral data about nucleic acids, and the associated experimental metadata, structural models, and links to literature. NACDDB covers CD data for various nucleic acid molecules, including DNA, RNA, DNA/RNA hybrids, and various nucleic acid derivatives. The entries are linked to primary sequence and experimental structural data, as well as to the literature.

Unraveling Membrane Perturbations Caused by the Bacterial Riboregulator Hfq.

Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNAs. Previously, we showed that the Hfq C-terminal region forms an amyloid-like structure and that these fibrils interact with membranes.

Evaluation of the Role of Bacterial Amyloid on Nucleoid Structure Using Cryo-Soft X-Ray Tomography.

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.

Analysis of Bacterial Amyloid Interaction with Lipidic Membrane by Orientated Circular Dichroism and Infrared Spectroscopies.

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.

Evaluation of Amyloid Inhibitor Efficiency to Block Bacterial Survival.

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.

Structural Information on Bacterial Amyloid and Amyloid-DNA Complex Obtained by Small-Angle Neutron or X-Ray Scattering.

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.