Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays-Protein and Buffer Composition Contribution.

ABSTRACT: 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.

SUBMITTER: Ruzza P

PROVIDER: S-EPMC8583428 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

UV-CD12: synchrotron radiation circular dichroism beamline at ANKA.

Project description:Synchrotron radiation circular dichroism (SRCD) is a rapidly growing technique for structure analysis of proteins and other chiral biomaterials. UV-CD12 is a high-flux SRCD beamline installed at the ANKA synchrotron, to which it had been transferred after the closure of the SRS Daresbury. The beamline covers an extended vacuum-UV to near-UV spectral range and has been open for users since October 2011. The current end-station allows for temperature-controlled steady-state SRCD spectroscopy, including routine automated thermal scans of microlitre volumes of water-soluble proteins down to 170?nm. It offers an excellent signal-to-noise ratio over the whole accessible spectral range. The technique of oriented circular dichroism (OCD) was recently implemented for determining the membrane alignment of ?-helical peptides and proteins in macroscopically oriented lipid bilayers as mimics of cellular membranes. It offers improved spectral quality <200?nm compared with an OCD setup adapted to a bench-top instrument, and accelerated data collection by a factor of ?3. In addition, it permits investigations of low hydrated protein films down to 130?nm using a rotatable sample cell that avoids linear dichroism artifacts.

| S-EPMC4416691 | biostudies-literature

Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism.

Project description:Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.

| S-EPMC6712486 | biostudies-literature

Going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy.

Project description:Circular dichroism (CD) spectroscopy is a fast, powerful, well-established, and widely used analytical technique in the biophysical and structural biology community to study protein secondary structure and to track changes in protein conformation in different environments. The use of the intense light of a synchrotron beam as the light source for collecting CD measurements has emerged as an enhanced method, known as synchrotron radiation circular dichroism (SRCD) spectroscopy, that has several advantages over the conventional CD method, including a significant spectral range extension for data collection, deeper access to the lower limit (cut-off) of conventional CD spectroscopy, an improved signal-to-noise ratio to increase accuracy in the measurements, and the possibility to collect measurements in highly absorbing solutions. In this review, we discuss different applications of the SRCD technique by researchers from Latin America. In this context, we specifically look at the use of this method for examining the secondary structure and conformational behavior of proteins belonging to the four main classes of the hierarchical protein domain classification CATH (Class, Architecture, Topology, Homology) database, focusing on the advantages and improvements associated with SRCD spectroscopy in terms of characterizing proteins composed of different structural elements.

| S-EPMC5662050 | biostudies-literature

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy.

Project description:We have used the combination of single-molecule Förster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Förster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a beta-structure content of the collapsed unfolded state of approximately 20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.

| S-EPMC1765419 | biostudies-literature

Synchrotron radiation circular dichroism spectroscopy reveals structural divergences in HDL-bound apoA-I variants.

Project description:Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) provides cardiovascular protection. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to analyze the dynamic solution structure of the apoA-I protein in the apo- and HDL-states and the protein structure conversion in HDL formation. Wild-type apoA-I protein was compared to human variants that either are protective (R173C, Milano) or lead to increased risk for ischaemic heart disease (A164S). Comparable secondary structure distributions in the HDL particles, including significant levels of beta strand/turn, were observed. ApoA-I Milano in HDL displayed larger size heterogeneity, increased protein flexibility, and an altered lipid-binding profile, whereas the apoA-I A164S in HDL showed decrease thermal stability, potentially linking the intrinsic HDL propensities of the variants to disease risk.

| S-EPMC5648894 | biostudies-other

Time resolved transient circular dichroism spectroscopy using synchrotron natural polarization.

Project description:Ultraviolet (UV) synchrotron radiation circular dichroism (SRCD) spectroscopy has made an important contribution to the determination and understanding of the structure of bio-molecules. In this paper, we report an innovative approach that we term time-resolved SRCD (tr-SRCD), which overcomes the limitations of current broadband UV SRCD setups. This technique allows accessing ultrafast time scales (down to nanoseconds), previously measurable only by other methods, such as infrared (IR), nuclear magnetic resonance (NMR), fluorescence and absorbance spectroscopies, and small angle X-ray scattering (SAXS). The tr-SRCD setup takes advantage of the natural polarization of the synchrotron radiation emitted by a bending magnet to record broadband UV CD faster than any current SRCD setup, improving the acquisition speed from 10 mHz to 130?Hz and the accessible temporal resolution by several orders of magnitude. We illustrate the new approach by following the isomer concentration changes of an azopeptide after a photoisomerization. This breakthrough in SRCD spectroscopy opens up a wide range of potential applications to the detailed characterization of biological processes, such as protein folding and protein-ligand binding.

| S-EPMC6823104 | biostudies-literature

Breaking the 200 nm limit for routine flow linear dichroism measurements using UV synchrotron radiation.

Project description:The first synchrotron radiation flow linear dichroism spectra are reported. High-quality spectral data can be collected from 450 nm down to 180 nm in contrast to the practical cutoff of approximately 200 nm on benchtop instruments. State-of-the-art microvolume capillary Couette flow linear dichroism was successfully ported to a synchrotron radiation source. The sample volume required is < 50 microL. A characterization of the synchrotron radiation linear dichroism with known DNA and DNA-ligand systems is presented and the viability of the setup confirmed. Typically, wavelengths down to 180 nm are now routinely accessible with a high signal/noise ratio with little limitation from the sample concentration. The 180 nm cutoff is due to the quartz of the Couette cell rather than the beamline itself. We show the application of the simultaneous determination of the sample absorption spectrum to calculate the reduced linear dichroism signal. Spectra for calf thymus DNA, DNA/ethidium bromide, and DNA/4',6-diamidino-2-phenylindole systems illustrate the quality of data that can be obtained.

| S-EPMC2599826 | biostudies-other

Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly.

Project description:Extramembranous domains play important roles in the structure and function of membrane proteins, contributing to protein stability, forming association domains, and binding ancillary subunits and ligands. However, these domains are generally flexible, making them difficult or unsuitable targets for obtaining high-resolution X-ray and NMR structural information. In this study we show that the highly sensitive method of synchrotron radiation circular dichroism (SRCD) spectroscopy can be used as a powerful tool to investigate the structure of the extramembranous C-terminal domain (CTD) of the prokaryotic voltage-gated sodium channel (Na(V)) from Bacillus halodurans, NaChBac. Sequence analyses predict its CTD will consist of an unordered region followed by an alpha-helix, which has a propensity to form a multimeric coiled-coil motif, and which could form an association domain in the homotetrameric NaChBac channel. By creating a number of shortened constructs we have shown experimentally that the CTD does indeed contain a stretch of approximately 20 alpha-helical residues preceded by a nonhelical region adjacent to the final transmembrane segment and that the efficiency of assembly of channels in the membrane progressively decreases as the CTD residues are removed. Analyses of the CTDs of 32 putative prokaryotic Na(V) sequences suggest that a CTD helical bundle is a structural feature conserved throughout the bacterial sodium channel family.

| S-EPMC2922593 | biostudies-literature

Investigating Membrane-Mediated Antimicrobial Peptide Interactions with Synchrotron Radiation Far-Infrared Spectroscopy.

Project description:Synchrotron radiation-based Fourier transform infrared spectroscopy enables access to vibrational information from mid over far infrared to even terahertz domains. This information may prove critical for the elucidation of fundamental bio-molecular phenomena including folding-mediated innate host defence mechanisms. Antimicrobial peptides (AMPs) represent one of such phenomena. These are major effector molecules of the innate immune system, which favour attack on microbial membranes. AMPs recognise and bind to the membranes whereupon they assemble into pores or channels destabilising the membranes leading to cell death. However, specific molecular interactions responsible for antimicrobial activities have yet to be fully understood. Herein we probe such interactions by assessing molecular specific variations in the near-THz 400-40 cm-1 range for defined helical AMP templates in reconstituted phospholipid membranes. In particular, we show that a temperature-dependent spectroscopic analysis, supported by 2D correlative tools, provides direct evidence for the membrane-induced and folding-mediated activity of AMPs. The far-FTIR study offers a direct and information-rich probe of membrane-related antimicrobial interactions.

| S-EPMC9303692 | biostudies-literature

Optical rotatory dispersion, circular dichroism and far-ultraviolet spectra of avidin and streptavidin.

Project description:1. The optical-rotatory-dispersion and circular-dichroism curves of avidin showed positive Cotton effects centred at 228mmu and 280mmu, close to the ultraviolet-absorption bands of tryptophan. These effects disappeared when avidin was dissociated into sub-units in guanidine hydrochloride. 2. Binding of biotin had only a small effect on the optical-rotatory-dispersion curve of avidin. 3. The absence of negative circular dichroism at wavelengths above 216mmu showed that there was little or no alpha-helix present in avidin. This interpretation was confirmed by Moffitt-Yang plots of the partial rotation due to the peptide bonds in the visible region of the spectrum. The calculated dispersion constants were remarkably similar to those of gamma-globulin and suggested the presence of peptide conformations other than alpha-helix and random coil. 4. The far-ultraviolet spectrum was also similar to that of gamma-globulin, the mean extinction coefficient of the peptide chromophore being much lower than the experimental value for a random-coil structure. 5. Streptavidin resembled avidin in showing two positive Cotton effects, but the negative dichroism below 220mmu suggested the presence of more alpha-helix than was found in avidin. Formation of the complex with biotin was accompanied by changes in rotation that were rather larger than those observed with avidin.

| S-EPMC1265192 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data