Search Results (7)

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

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

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

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

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

Many neurodegenerative diseases, including Huntington’s, Alzheimer’s and Parkinson’s diseases, are characterized by protein misfolding and aggregation. The capability of trehalose to interfere with protein misfolding and aggregation has been recently evaluated by several research groups. In the present work, we studied, by means of synchrotron radiation circular dichroism (SRCD) spectroscopy, the dose-effect of trehalose on α-synuclein conformation and/or stability to probe the capability of this osmolyte to interfere with α-synuclein’s aggregation. Our study indicated that a low trehalose concentration stabilized α-synuclein folding much better than at high concentration by blocking in vitro α-synuclein’s polymerisation. These results suggested that trehalose could be associated with other drugs leading to a new approach for treating Parkinson’s and other brain-related diseases. Full article