Synchrotron Radiation Circular Dichroism Research Articles

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. To fulfill the growing need to investigate nano-bio interactions, this study provides a systematic understanding of two key aspects: (i) protein corona (PC) formation and (ii) NP-induced modifications on protein's structure and stability. A methodology was developed by combining orthogonal techniques to analyze both quantitative and qualitative aspects of nano-bio interactions, using human serum albumin (HSA) as a model protein. Protein quantification via liquid chromatography-mass spectrometry, and capillary zone electrophoresis (CZE) clarified adsorbed protein quantity and stability. CZE further unveiled qualitative insights into HSA forms (native, glycated HSA and cysteinylated), while synchrotron radiation circular dichroism enabled analyzing HSA's secondary structure and thermal stability. Comparative investigations of NP cores (organic vs. hybrid), and shells (with or without polyethylene glycol (PEG)) revealed pivotal factors influencing nano-bio interactions. Polymeric NPs based on poly(lactic-co-glycolic acid) (PLGA) and hybrid NPs based on metal-organic frameworks (nanoMOFs) presented distinct HSA adsorption profiles. PLGA NPs had protein-repelling properties while inducing structural modifications on HSA. In contrast, HSA exhibited a high affinity for nanoMOFs forming a PC altering thereby the protein structure. A shielding effect was gained through PEGylation for both types of NPs, avoiding the PC formation as well as the alteration of unbound HSA structure.

Pea and soy protein isolate fractal gels: The role of protein composition, structure and solubility on their gelation behaviour

The gelation behaviour of two different pea protein isolates and one soy protein isolate were investigated with a focus on the role of the protein properties. Protein solubility was the lowest in pH 3 citrate-phosphate buffer (<10% w/w), increased in pH 7.4 phosphate-buffered saline (12–21% w/w), and was the highest in pH 7.6 MilliQ water (∼20–40% w/w). Heat-induced gelation conditions for the protein sources were sensitive to both the soluble and the insoluble fractions as obtained during extraction. At low protein concentrations (≤5% w/v), the proteins started to lose their viscoelastic behaviour and exhibited predominantly viscous properties. Fitting of the fractional Kelvin-Voigt model to the frequency sweeps showed an increase in the fractal gel strength with increasing protein concentration. Secondary structures of the soluble species showed mostly unordered proteins, suggesting that the proteins were denatured during the commercial extraction process although gelation has to date been suggested to be highly dependent on the denaturation of soluble proteins. Synchrotron Radiation Circular Dichroism measurements of the insoluble proteins showed a significant amount of ordered protein structures. SEM imaging of the gels also suggested a new gelation pathway in which insoluble proteins act as dispersed fillers within a continuous matrix of soluble proteins. This research elucidated the role of different protein fractions, globulins and albumins, and their secondary structure in the formation of a gel network and how this affects their viscoelastic behaviour.

KCD: A prediction web server of knowledge-based circular dichroism.

We present a web server that predicts the far-UV circular dichroism (CD) spectra of proteins by utilizing their three-dimensional (3D) structures from the Protein Data Bank (PDB). The main algorithm is based on the classical theory of optical activity together with a set of atomic complex polarizabilities, which are obtained from the analysis of a series of synchrotron radiation CD spectra and their related 3D structures from the PDB. The results of our knowledge-based CD method (KCD) are in good agreement with measured spectra that could include the effect of D-amino acids. Our method also delivers some of the most accurate predictions, in comparison with the calculated spectra from well-established models. Specifically, using a metric of closeness based on normalized absolute deviations between experimental and calculated spectra, the mean values for a series of 57 test proteins give the following figures for such models: 0.26 KCD, 0.27 PDBMD2CD, 0.30 SESCA, and 0.47 DichroCalc. From another point of view, it is worth mentioning the remarkable capabilities of the recent approaches based on artificial intelligence, which can precisely predict the native structure of proteins. The structure of proteins, however, is flexible and can be modified by a diversity of environmental factors such as interactions with other molecules, mechanical stresses, variations of temperature, pH, or ionic strength. Experimental CD spectra together with reliable predictions can be utilized to assess eventual secondary structural changes. A similar kind of evaluation can be done for the case of an incomplete protein structure that has been reconstructed by using different approaches. The KCD method can be freely accessed from: https://kcd.cinvestav.mx/.

Human Serum Albumin in the Presence of Small Platinum Nanoparticles

Noble metal materials, especially platinum nanoparticles (Pt NPs), have immense potential in nanomedicine as therapeutic agents on account of their high electron density and their high surface area. Intravenous injection is proposed as the best mode to deliver the product to patients. However, our understanding of the reaction of nanoparticles with blood components, especially proteins, is far behind the explosive development of these agents. Using synchrotron radiation circular dichroism (SRCD), we investigated the structural and stability changes of human serum albumin (HSA) upon interaction with PEG-OH coated Pt NPs at nanomolar concentrations, conditions potentially encountered for intravenous injection. There is no strong complexation found between HSA and Pt NPs. However, for the highest molar ratio of NP:HSA of 1:1, an increase of 18 °C in the thermal unfolding of HSA was observed, which is attributed to increased thermal stability of HSA generated by preferential hydration. This work proposes a new and fast method to probe the potential toxicity of nanoparticles intended for clinical use with intravenous injection.

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 Klebsiella pneumoniae 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. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the β-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.

Structural properties of the HNF-1A transactivation domain.

Hepatocyte nuclear factor 1α (HNF-1A) is a transcription factor with important gene regulatory roles in pancreatic β-cells. HNF1A gene variants are associated with a monogenic form of diabetes (HNF1A-MODY) or an increased risk for type 2 diabetes. While several pancreatic target genes of HNF-1A have been described, a lack of knowledge regarding the structure-function relationships in HNF-1A prohibits a detailed understanding of HNF-1A-mediated gene transcription, which is important for precision medicine and improved patient care. Therefore, we aimed to characterize the understudied transactivation domain (TAD) of HNF-1A in vitro. We present a bioinformatic approach to dissect the TAD sequence, analyzing protein structure, sequence composition, sequence conservation, and the existence of protein interaction motifs. Moreover, we developed the first protocol for the recombinant expression and purification of the HNF-1A TAD. Small-angle X-ray scattering and synchrotron radiation circular dichroism suggested a disordered conformation for the TAD. Furthermore, we present functional data on HNF-1A undergoing liquid-liquid phase separation, which is in line with in silico predictions and may be of biological relevance for gene transcriptional processes in pancreatic β-cells.

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)9 and a mixed 18-mer double stranded DNA at 1, 5, and 10 mM in 100 mM or 4 M NaCl. SRCD of low molecular weight salmon DNA was also measured at a 10 mg/ml concentration. These results represent the first report of CD spectra of DNA samples measured at concentrations comparable to those found in the nucleus. The results suggest that dsDNA maintain very similar structures at concentrations up to tens of mg/ml, as evident by the very similar CD patterns in this concentration range. Furthermore, the SRCD allowed for the recording of CD patterns of DNA in the far UV region, which is not readily accessible by standard benchtop CD spectropolarimeters. These far UV signals appear to be quite characteristic of DNA structures and are sensitive to sample conditions.

Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate.

Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.

Antimicrobial Activity of an Fmoc-Plantaricin 149 Derivative Peptide against Multidrug-Resistant Bacteria.

Antimicrobial resistance poses a major threat to public health. Given the paucity of novel antimicrobials to treat resistant infections, the emergence of multidrug-resistant bacteria renewed interest in antimicrobial peptides as potential therapeutics. This study designed a new analog of the antimicrobial peptide Plantaricin 149 (Pln149-PEP20) based on previous Fmoc-peptides. The minimal inhibitory concentrations of Pln149-PEP20 were determined for 60 bacteria of different species and resistance profiles, ranging from 1 mg/L to 128 mg/L for Gram-positive bacteria and 16 to 512 mg/L for Gram-negative. Furthermore, Pln149-PEP20 demonstrated excellent bactericidal activity within one hour. To determine the propensity to develop resistance to Pln149-PEP20, a directed-evolution in vitro experiment was performed. Whole-genome sequencing of selected mutants with increased MICs and wild-type isolates revealed that most mutations were concentrated in genes associated with membrane metabolism, indicating the most likely target of Pln149-PEP20. Synchrotron radiation circular dichroism showed how this molecule disturbs the membranes, suggesting a carpet mode of interaction. Membrane depolarization and transmission electron microscopy assays supported these two hypotheses, although a secondary intracellular mechanism of action is possible. The molecule studied in this research has the potential to be used as a novel antimicrobial therapy, although further modifications and optimization remain possible.

The PCDDB project: Tools and resources for CD analyses, archiving, display, and sharing.

The Protein Circular Dichroism Data Bank Project was initiated more than 20 years ago as a means of creating an integrated set of software and tools to aid in analyses, interpretations, visual displays, and data sharing of circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD) spectra and metadata. It has been a basis for the creation of a wide range of novel biophysical and computational methods, and has resulted from the coordinated effort of our two labs and many collaborators. At its heart has been the DichroWeb analysis server, which has been used for ∼2 M secondary structure analyses by >9000 users, and which has included the creation of new computational methods and associated reference databases for examining soluble, membrane, and intrinsically-disordered proteins. The PCDDB itself was created as a repository for CD data and metadata with links to a wide range of bioinformatics, computational, and structural databases and tools, encouraging interoperability of CD and other biophysical methods. It includes links to primary sequence (UniProt) and structural (PDB) databases, as well as to secondary databases such as the Enzyme Commission functional database and the CATH fold database. In addition, tools were created for data processing, and display of CD spectra, for validation of spectral quality (ValiDichro), for integration with other methods such as PDB2CD, PDBMD2CD, 2StructCompare. and DichroCompare, plus novel methods of visualisation, including kinetics of protein folding. Software created is available online via GitHub. In addition to providing widely-used research tools, this project has also been the core teaching tool for CD summer schools and courses worldwide, and has benefitted from the contributions of many biophysicis groups worldwide. [Supported by grants from the UK Biotechnology and Biological Resources programme of the BBSRC).

Mechanism of protein stabilization in crowded solution and preservation in desiccated sugar glass.

The highly crowded cellular milieu includes countless different macromolecules and osmolytes aimed at regulating osmotic stress while protecting proteins and other macromolecules. Of special interest is the formation of cellular glassy matrices composed of sugars, which strongly correlate with the ability of some yeast, nematodes, and tardigrades to survive desiccation and extreme temperature changes. Remarkably, these carbohydrates stabilize proteins in crowded solutions and preserve them in the dry glassy state. Yet, although considerable efforts have been directed to discern how crowding stabilizes proteins, the molecular-level stabilization mechanism has remained elusive. We have followed the effect of various osmolytes on the native state stability of model miniproteins in crowded solutions using circular dichroism (CD) spectroscopy and our newly developed mean-field theory for crowding. We show that protein native state stability and folding thermodynamic signature can be explained using three distinct types of interactions: excluded volume, solvent-osmolyte non-ideal mixing, and osmolyte-protein soft-interactions. Furthermore, using our combination of synchrotron radiation CD spectroscopy together with sampling enhanced molecular dynamics (MD) simulations we have been able to shed light on the stability and new emerging nanostructures of proteins when they are embedded in sugar glass.

Controlled modulation of the dynamics of the Deinococcus grandis Dps N-terminal tails by divalent metals.

DNA-binding proteins from starved cells (Dps) are small multifunctional nanocages expressed by prokaryotes in acute oxidative stress conditions or during the starvation-induced stationary phase, as a bacterial defense mechanism. Dps proteins protect bacterial DNA from damage by either direct binding or by removing precursors of reactive oxygen species from solution. The DNA-binding properties of most Dps proteins studied so far are related to their unordered, flexible, N- and C-terminal extensions. In a previous work, we revealed that the N-terminal tails of Deinoccocus grandis Dps shift from an extended to a compact conformation depending on the ionic strength of the buffer and detected a novel high-spin ferrous iron center in the proximal ends of those tails. In this work, we further explore the conformational dynamics of the protein by probing the effect of divalent metals binding to the tail by comparing the metal-binding properties of the wild-type protein with a binding site-impaired D34A variant using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism, and small-angle X-ray scattering. The N-terminal ferrous species was also characterized by Mössbauer spectroscopy. The results herein presented reveal that the conformation of the N-terminal tails is altered upon metal binding in a gradual, reversible, and specific manner. These observations may point towards the existence of a regulatory process for the DNA-binding properties of Dps proteins through metal binding to their N- and/or C-terminal extensions.

Synchrotron radiation circular dichroism spectroscopy reveals that gold and silver nanoparticles modify the secondary structure of a lung surfactant protein B analogue.

Inhaled nanoparticles (NPs) depositing in the alveolar region of the lung interact initially with a surfactant layer and in vitro studies have demonstrated that NPs can adversely affect the biophysical function of model pulmonary surfactants (PS), of which surfactant protein B (SP-B) is a key component. Other studies have demonstrated the potential for NPs to modify the structure and function of proteins. It was therefore hypothesised that NPs may affect the biophysical function of PS by modifying the structure of SP-B. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to explore the effect of various concentrations of gold nanoparticles (AuNPs) (5, 10, 20 nm), silver nanoparticles (AgNPs) (10 nm) and silver citrate on the secondary structure of surfactant protein B analogue, SP-B1-25, in a TFE/PB dispersion. For Au and Ag NPs the SRCD spectra indicated a concentration dependent reduction in the α-helical structure of SP-B1-25 (5 nm AuNP ≈ 10 nm AgNP ≫ 10 nm AuNP > 20 nm AuNP). For AuNPs the effect was greater for the 5 nm size, which was not fully explained by consideration of surface area. The impact of the 10 nm AgNPs was greater than that of the 10 nm AuNPs and the effect of AgNPs was greater than that of silver citrate at equivalent Ag mass concentrations. For 10 nm AuNPs, SRCD spectra for dispersions in, the more physiologically relevant, DPPC showed a similar concentration dependent pattern. The results demonstrate the potential for inhaled NPs to modify SP-B1-25 structure and thus potentially adversely impact the physiological function of the lung, however, further studies are necessary to confirm this.

The Apparent Organ-Specificity of Amyloidogenic ApoA-I Variants Is Linked to Tissue-Specific Extracellular Matrix Components

Apolipoprotein A-I (ApoA-I) amyloidosis is a rare protein misfolding disease where fibrils of the N-terminal domain of the protein accumulate in several organs, leading to their failure. Although ApoA-I amyloidosis is systemic, the different amyloidogenic variants show a preferential tissue accumulation that appears to correlate with the location of the mutation in the protein sequence and with the local extracellular microenvironment. However, the factors leading to cell/tissues damage, as well as the mechanisms behind the observed organ specificity are mostly unknown. Therefore, we investigated the impact of ApoA-I variants on cell physiology and the mechanisms driving the observed tissue specificity. We focused on four ApoA-I amyloidogenic variants and analyzed their cytotoxicity as well as their ability to alter redox homeostasis in cell lines from different tissues (liver, kidney, heart, skin). Moreover, variant-specific interactions with extracellular matrix (ECM) components were measured by synchrotron radiation circular dichroism and enzyme-linked immunosorbent assay. Data indicated that ApoA-I variants exerted a cytotoxic effect in a time and cell-type-specific manner that seems to be due to protein accumulation in lysosomes. Interestingly, the ApoA-I variants exhibited specific preferential binding to the ECM components, reflecting their tissue accumulation pattern in vivo. While the binding did not to appear to affect protein conformations in solution, extended incubation of the amyloidogenic variants in the presence of different ECM components resulted in different aggregation propensity and aggregation patterns.

Length matters: Functional flip of the short TatA transmembrane helix

The twin arginine translocase (Tat) exports folded proteins across bacterial membranes. The putative pore-forming or membrane-weakening component (TatAd in B. subtilis) is anchored to the lipid bilayer via an unusually short transmembrane α-helix (TMH), with less than 16 residues. Its tilt angle in different membranes was analyzed under hydrophobic mismatch conditions, using synchrotron radiation circular dichroism and solid-state NMR. Positive mismatch (introduced either by reconstitution in short-chain lipids or by extending the hydrophobic TMH length) increased the helix tilt of the TMH as expected. Negative mismatch (introduced either by reconstitution in long-chain lipids or by shortening the TMH), on the other hand, led to protein aggregation. These data suggest that the TMH of TatA is just about long enough for stable membrane insertion. At the same time, its short length is a crucial factor for successful translocation, as demonstrated here in native membrane vesicles using an in vitro translocation assay. Furthermore, when reconstituted in model membranes with negative spontaneous curvature, the TMH was found to be aligned parallel to the membrane surface. This intrinsic ability of TatA to flip out of the membrane core thus seems to play a key role in its membrane-destabilizing effect during Tat-dependent translocation.

Modulating the poly-l-lysine structure through the control of the protonation–deprotonation state of l-lysine

Designing the architecture of l-lysine-based polymeric structures is a highly challenging task that requires careful control of the amino acid reactive groups. Conventional processes to obtain branched polylysine need several steps and the addition of specific catalysts. In the present work, to gain a better understanding and control of the formation of l-lysine-based polymers, we have investigated the correlation between the protonation state of l-lysine and the corresponding hydrothermally grown structures. The samples have been characterized by combining optical spectroscopies, such as UV–Vis, fluorescence, and synchrotron radiation circular dichroism with structural analysis by Nuclear Magnetic Resonance, Fourier Transform Infrared spectroscopy, and dynamic light scattering. We have observed that aqueous precursor solutions with alkaline pHs promote the formation of branched structures. In contrast, high pHs favour the reactivity of the ε-amino groups leading to linear structures, as shown by circular dichroism analyses. On the other hand, acidic conditions trigger the branching of the amino acid. Interestingly, the polymeric forms of l-lysine emit in the blue because the increasing number of intermolecular hydrogen bonds promote the intermolecular charge transfer responsible for the emission. Understanding the correlation between the l-lysine charged states and the polymeric structures that could form controlling the protonation–deprotonation states of the amino acid opens the route to a refined design of polypeptide systems based on l-lysine.

Molecular dynamics simulations of the adsorption of an intrinsically disordered protein: Force field and water model evaluation in comparison with experiments.

This study investigates possible structural changes of an intrinsically disordered protein (IDP) when it adsorbs to a solid surface. Experiments on IDPs primarily result in ensemble averages due to their high dynamics. Therefore, molecular dynamics (MD) simulations are crucial for obtaining more detailed information on the atomistic and molecular levels. An evaluation of seven different force field and water model combinations have been applied: (A) CHARMM36IDPSFF + CHARMM-modified TIP3P, (B) CHARMM36IDPSFF + TIP4P-D, (C) CHARMM36m + CHARMM-modified TIP3P, (D) AMBER99SB-ILDN + TIP3P, (E) AMBER99SB-ILDN + TIP4P-D, (F) AMBERff03ws + TIP4P/2005, and (G) AMBER99SB-disp + disp-water. The results have been qualitatively compared with those of small-angle X-ray scattering, synchrotron radiation circular dichroism spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy. The model IDP corresponds to the first 33 amino acids of the N-terminal of the magnesium transporter A (MgtA) and is denoted as KEIF. With a net charge of +3, KEIF is found to adsorb to the anionic synthetic clay mineral Laponite® due to the increase in entropy from the concomitant release of counterions from the surface. The experimental results show that the peptide is largely disordered with a random coil conformation, whereas the helical content (α- and/or 310-helices) increased upon adsorption. MD simulations corroborate these findings and further reveal an increase in polyproline II helices and an extension of the peptide conformation in the adsorbed state. In addition, the simulations provided atomistic resolution of the adsorbed ensemble of structures, where the arginine residues had a high propensity to form hydrogen bonds with the surface. Simulations B, E, and G showed significantly better agreement with experiments than the other simulations. Particularly noteworthy is the discovery that B and E with TIP4P-D water had superior performance to their corresponding simulations A and D with TIP3P-type water. Thus, this study shows the importance of the water model when simulating IDPs and has also provided an insight into the structural changes of surface-active IDPs induced by adsorption, which may play an important role in their function.

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. Additionally, for all entries, 3D structure models are provided. All entries undergo expert validation and curation procedures to ensure completeness, consistency, and quality of the data included. The NACDDB is open for submission of the CD data for nucleic acids. NACDDB is available at: https://genesilico.pl/nacddb/.

Adjuvant effect of mesoporous silica SBA-15 on anti-diphtheria and anti-tetanus humoral immune response

Routine immunization against diphtheria and tetanus has drastically reduced the incidence of these diseases worldwide. Anti-diphtheria/tetanus vaccine has in general aluminum salt as adjuvant in its formulation that can produce several adverse effects. There is a growing interest in developing new adjuvants. In this study, we evaluated the efficiency of SBA-15 as an adjuvant in subcutaneous immunization in mice with diphtheria (dANA) and tetanus (tANA) anatoxins as well as with the mixture of them (dtANA). The tANA molecules and their encapsulation in SBA-15 were characterized using Small-Angle X-ray Scattering (SAXS), Dynamical Light Scattering (DLS), Nitrogen Adsorption Isotherm (NAI), Conventional Circular Dichroism (CD)/Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy, and Tryptophan Fluorescence Spectroscopy (FS). The primary and secondary antibody response elicited by subcutaneous immunization of High (HIII) and Low (LIII) antibody responder mice with dANA, tANA, or dtANA encapsulated in the SBA-15 were determined. We demonstrated that SBA-15 increases the immunogenicity of dANA and tANA antigens, especially when administered in combination. We also verified that SBA-15 modulates the antibody response of LIII mice, turning them into high antibody responder. Thus, these results suggest that SBA-15 may be an effective adjuvant for different vaccine formulations.

Critical structural elements for the antigenicity of wheat allergen LTP1 (Tri a 14) revealed by site-directed mutagenesis

Lipid transfer proteins (LTPs) were identified as allergens in a large variety of pollens and foods, including cereals. LTPs belong to the prolamin superfamily and display an α-helical fold, with a bundle of four α-helices held together by four disulfide bonds. Wheat LTP1 is involved in allergic reactions to food. To identify critical structural elements of antibody binding to wheat LTP1, we used site-directed mutagenesis on wheat recombinant LTP1 to target: (i) sequence conservation and/or structure flexibility or (ii) each disulfide bond. We evaluated the modifications induced by these mutations on LTP1 secondary structure by synchrotron radiation circular dichroism and on its antigenicity with patient’s sera and with mouse monoclonal antibodies. Disruption of the C28–C73 disulfide bond significantly affected IgE-binding and caused protein denaturation, while removing C13–C27 bond decreased LTP1 antigenicity and slightly modified LTP1 overall folding. In addition, we showed Lys72 to be a key residue; the K72A mutation did not affect global folding but modified the local 3D structure of LTP1 and strongly reduced IgE-binding. This work revealed a cluster of residues (C13, C27, C28, C73 and K72), four of which embedded in disulfide bonds, which play a critical role in LTP1 antigenicity.