Search Results (17)

Circular dichroism (CD) spectroscopy has emerged as a powerful tool in the study of protein folding, structure, and function. This review explores the versatile applications of CD spectroscopy in unraveling the intricate relationship between protein conformation and biological activity. A key advantage of CD spectroscopy is its ability to analyze protein samples with minimal quantity requirements, making it an attractive technique for studying proteins that are scarce or difficult to produce. Moreover, CD spectroscopy enables the monitoring of physical and chemical environmental effects on protein structures, providing valuable insights into the dynamic behavior of proteins in different conditions. In recent years, the use of synchrotron radiation as a light source for CD measurements has gained traction, offering enhanced sensitivity and resolution. By combining the advantages of CD spectroscopy, such as minimal sample requirements and the ability to probe environmental effects, with the emerging capabilities of synchrotron radiation (SRCD), researchers have an unprecedented opportunity to explore the diverse aspects of protein behavior. This review highlights the significance of CD spectroscopy in protein research and the growing role of synchrotron radiation in advancing our understanding of protein behavior, aiming to provide novel insights and applications in various fields, including drug discovery, protein engineering, and biotechnology. A brief overview of Solid-State Circular Dichroism (SSCD) is also included. Full article

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. Full article

Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The Myxococcus xanthus encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA−pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein (T_m) slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo. Full article

The aim of this work was to test polyamines as potential natural substrates of the Acinetobacter baumannii chlorhexidine efflux protein AceI using near-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. The Gram-negative bacterium A. Baumannii is a leading cause of hospital-acquired infections and an important foodborne pathogen. A. Baumannii strains are becoming increasingly resistant to antimicrobial agents, including the synthetic antiseptic chlorhexidine. AceI (144-residues) was the founding member of the recently recognised PACE family of bacterial multidrug efflux proteins. Using the plasmid construct pTTQ18-aceI(His₆) containing the A. baumannii aceI gene directly upstream from a His₆-tag coding sequence, expression of AceI(His₆) was amplified in E. coli BL21(DE3) cells. Near-UV (250–340 nm) SRCD measurements were performed on detergent-solubilised and purified AceI(His₆) at 20 °C. Sample and SRCD experimental conditions were identified that detected binding of the triamine spermidine to AceI(His₆). In a titration with spermidine (0–10 mM), this binding was saturable and fitting of the curve for the change in signal intensity produced an apparent binding affinity (K_D) of 3.97 ± 0.45 mM. These SRCD results were the first experimental evidence obtained for polyamines as natural substrates of PACE proteins. Full article

The tau protein, a soluble protein associated with microtubules, which is involved in the assembly and stabilization of cytoskeletal elements, was found to form neurofibrillary tangles in different neurodegenerative diseases. Insoluble tau aggregates were observed to be organized in paired helical filaments (PHFs) and straight filaments (SFs). Recently, two small sequences (306–311 and 275–280) in the microtubule-binding region (MTBR), named PHF6 and PHF6*, respectively, were found to be essential for tau aggregation. Since a possible therapeutic approach consists of impairing amyloid formation either by stabilizing the native proteins or reducing the level of amyloid precursors, here we use synchrotron radiation circular dichroism (SRCD) at Diamond B23 beamline to evaluate the inhibitory effects of two small molecules, trehalose and ceftriaxone, against the aggregation of a small peptide containing the PHF6* sequence. Our results indicate that both these molecules, ceftriaxone and trehalose, increased the stability of the peptide toward aggregation, in particular that induced by heparin. With trehalose being present in many fruits, vegetables, algae and processed foods, these results support the need to investigate whether a diet richer in trehalose might exert a protective effect toward pathologies linked to protein misfolding. Full article

Hfq is a bacterial master regulator which promotes the pairing of nucleic acids. Due to the high molecular weight of the complexes formed between nucleic acids and the amyloid form of the protein, it is difficult to analyze solely by a gel shift assay the complexes formed, as they all migrate at the same position in the gel. In addition, precise kinetics measurements are not possible using a gel shift assay. Here, we used a synchrotron-based biophysical approach, synchrotron radiation circular dichroism (SRCD), to probe the interaction of the Escherichia coli Hfq C-terminal amyloid region with nucleic acids involved in the control of ColE1-like plasmid replication. We observed that this C-terminal region of Hfq has an unexpected and significant effect on the annealing of nucleic acids involved in this process and, more importantly, on their alignment. Functional consequences of this newly discovered property of the Hfq amyloid region are discussed in terms of the biological significance of Hfq in the ColE1-type plasmid replication process and antibiotic resistance. Full article

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate. Full article

Hfq is a bacterial RNA chaperone which promotes the pairing of small noncoding RNAs to target mRNAs, allowing post-transcriptional regulation. This RNA annealing activity has been attributed for years to the N-terminal region of the protein that forms a toroidal structure with a typical Sm-fold. Nevertheless, many Hfqs, including that of Escherichia coli, have a C-terminal region with unclear functions. Here we use a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to probe the interaction of the E. coli Hfq C-terminal amyloid region with RNA and its effect on RNA annealing. This C-terminal region of Hfq, which has been described to be dispensable for sRNA:mRNA annealing, has an unexpected and significant effect on this activity. The functional consequences of this novel property of the amyloid region of Hfq in relation to physiological stress are discussed. Full article

Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180–250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption. Full article

Food processing affects the structure and chemical state of proteins. In particular, protein oxidation occurs and may impair protein properties. These chemical reactions initiated during processing can develop during digestion. Indeed, the physicochemical conditions of the stomach (oxygen pressure, low pH) favor oxidation. In that respect, digestive proteases may be affected as well. Yet, very little is known about the link between endogenous oxidation of digestive enzymes, their potential denaturation, and, therefore, food protein digestibility. Thus, the objective of this study is to understand how oxidative chemical processes will impact the pepsin secondary structure and its hydrolytic activity. The folding and unfolding kinetics of pepsin under oxidative conditions was determined using Synchrotron Radiation Circular Dichroism. SRCD gave us the possibility to monitor the rapid kinetics of protein folding and unfolding in real-time, giving highly resolved spectral data. The proteolytic activity of control and oxidized pepsin was investigated by MALDI-TOF mass spectrometry on a meat protein model, the creatine kinase. MALDI-TOF MS allowed a rapid evaluation of the proteolytic activity through peptide fingerprint. This study opens up new perspectives by shifting the digestion paradigm taking into account the gastric digestive enzyme and its substrate. Full article

Insulin mucoadhesive buccal films (MBF) are a noninvasive insulin delivery system that offers an advantageous alternative route of administration to subcutaneous injection. One major concern in the formulation of insulin MBF is the preservation of an insulin secondary structure in the presence of the other film components. Buccal films were formulated using chitosan, glycerin, and L-arginine. The MBF-forming solutions (MBF-FS) and the films (MBF) were examined for their chemical and structural stability and for their in vivo activity. Enzyme-Linked Immunosorbent Assay (ELISA) of the insulin-loaded MBF showed that each individualized unit dose was at least loaded with 80% of the insulin theoretical dose. Results of Synchrotron Radiation Circular Dichroism (SRCD) measurements revealed that MBF-FS retained the α-helices and β–sheets conformations of insulin. Fourier transform infrared (FTIR)-microspectroscopy (FTIR-MS) examination of insulin MBF revealed the protective action of L-arginine on insulin structure by interacting with chitosan and minimizing the formation of an unordered structure and β-strand. A blood glucose-lowering effect of insulin MBF was observed in comparison with subcutaneous (S.C) injection using a rat model. As a result; chitosan-based MBFs were formulated and characterized using SRCD and FTIR-MS techniques. Furthermore, the results of in vivo testing suggested the MBFs as a promising delivery system for insulin. Full article

The novel vertical sample chamber, developed at the B23 beamline for synchrotron radiation circular dichroism (SRCD), has enabled the Diamond User community to conduct different types of experiments from high throughput CD of protein and DNA folding using 96-well multiplates to CD imaging at high spatial resolution. Here, we present the application of CD imaging to large areas of achiral polymer PVA films doped with D-dopa to assess the chiral homogeneity of the film preparation with potential antimicrobial property. Synopsis: CDi application of Diamond B23 SRCD beamline. Full article

Polyphenols are an important constituent of wines and they are largely studied due to their antioxidant properties and for their effects on wine quality and stability, which is also related to their capacity to bind to proteins. The effects of some selected polyphenols, including procyanidins B1 and B2, tannic acid, quercetin, and rutin, as well as those of a total white wine procyanidin extract on the conformational properties of the major wine protein VVTL1 (Vitis vinifera Thaumatin-Like-1) were investigated by Synchrotron Radiation Circular Dichroism (SRCD). Results showed that VVTL1 interacts with polyphenols as demonstrated by the changes in the secondary (far-UV) and tertiary (near-UV) structures, which were differently affected by different polyphenols. Additionally, polyphenols modified the two melting temperatures (T_M) that were found for VVTL1 (32.2 °C and 53.9 °C for the protein alone). The circular dichroism (CD) spectra in the near-UV region revealed an involvement of the aromatic side-chains of the protein in the interaction with phenolics. The data demonstrate the existence of an interaction between polyphenols and VVTL1, which results in modification of its thermal and UV denaturation pattern. This information can be useful in understanding the behavior of wine proteins in presence of polyphenols, thus giving new insights on the phenomena that are involved in wine stability. Full article

Double-strand breaks of DNA may lead to discontinuous DNA and consequent loss of genetic information, which may result in mutations or, ultimately, carcinogenesis. To avoid such potentially serious situations, cells have evolved efficient DNA damage repair systems. It is thought that DNA-repair processes involve drastic alterations of chromatin and histone structures, but detection of these altered structures in DNA-damaged cells remains rare in the literature. Recently, synchrotron radiation circular dichroism (SRCD) spectroscopy, which can provide secondary structural information of proteins in solution, has identified structural alterations of histone proteins induced by DNA damage responses. In this review, these results and experimental procedures are discussed with the aim of facilitating further studies of the chromatin remodeling and DNA damage repair pathways using SRCD spectroscopy. Full article

The Hfq protein is reported to be involved in environmental adaptation and virulence of several bacteria. In Gram-negative bacteria, Hfq mediates the interaction between regulatory noncoding RNAs and their target mRNAs. Besides these RNA-related functions, Hfq is also associated with DNA and is a part of the bacterial chromatin. Its precise role in DNA structuration is, however, unclear and whether Hfq plays a direct role in DNA-related processes such as replication or recombination is controversial. In previous works, we showed that Escherichia coli Hfq, or more precisely its amyloid-like C-terminal region (CTR), induces DNA compaction into a condensed form. In this paper, we evidence a new property for Hfq; precisely we show that its CTR influences double helix structure and base tilting, resulting in a strong local alignment of nucleoprotein Hfq:DNA fibers. The significance of this alignment is discussed in terms of chromatin structuration and possible functional consequences on evolutionary processes and adaptation to environment. Full article