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Journal of Mass Spectrometry : JMS Dec 2010Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein... (Review)
Review
Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposure of proteins to a 'white' X-ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal-to-noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis-driven structural mass spectrometry experiments.
Topics: Hydroxyl Radical; Mass Spectrometry; Peptide Mapping; Protein Conformation; Proteins
PubMed: 20812376
DOI: 10.1002/jms.1808 -
Journal of the American Chemical Society May 2023Lytic polysaccharide monooxygenases have received significant attention as catalytic convertors of biomass to biofuel. Recent studies suggest that its peroxygenase...
Lytic polysaccharide monooxygenases have received significant attention as catalytic convertors of biomass to biofuel. Recent studies suggest that its peroxygenase activity (i.e., using HO as an oxidant) is more important than its monooxygenase functionality. Here, we describe new insights into peroxygenase activity, with a copper(I) complex reacting with HO leading to site-specific ligand-substrate C-H hydroxylation. [Cu(TMGtren)] () (TMGtren = 1,1,1-Tris{2-[-(1,1,3,3-tetramethylguanidino)]ethyl}amine) and a dry source of hydrogen peroxide, (-TolP═O·HO) react in the stoichiometry, [Cu(TMGtren)] + HO → [Cu(TMGtren-OH)] + HO, wherein a ligand -methyl group undergoes hydroxylation giving TMGtren-OH. Furthermore, Fenton-type chemistry (Cu + HO → Cu-OH + ·OH) is displayed, in which (i) a Cu(II)-OH complex could be detected during the reaction and it could be separately isolated and characterized crystallographically and (ii) hydroxyl radical (·OH) scavengers either quenched the ligand hydroxylation reaction and/or (iii) captured the ·OH produced.
Topics: Copper; Hydrogen Peroxide; Hydroxylation; Ligands; Mixed Function Oxygenases; Hydroxyl Radical; Oxidation-Reduction
PubMed: 37195014
DOI: 10.1021/jacs.3c02273 -
Microbiology Spectrum Aug 2022Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common drug-resistant bacteria and poses a significant threat to human health. Due to the emergence...
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common drug-resistant bacteria and poses a significant threat to human health. Due to the emergence of multidrug resistance, limited drugs are available for the treatment of MRSA infections. In recent years, platelets have been reported to play important roles in inflammation and immune responses, in addition to their functions in blood hemostasis and clotting. We and other researchers have previously reported that platelets can inhibit Staphylococcus aureus growth. However, it remained unclear whether platelets have the same antibacterial effect on drug-resistant strains. In this study, we hypothesized that platelets may also inhibit the growth of MRSA; the results confirmed that platelets significantly inhibited the growth of MRSA . In a murine model of MRSA infection, we found that a platelet transfusion alleviated the symptoms of MRSA infection; in contrast, depletion of platelets aggravated infective symptoms. Moreover, we observed an overproduction of hydroxyl radicals in MRSA following platelet treatment, which induced apoptosis-like death of MRSA. Our findings demonstrate that platelets can inhibit MRSA growth by promoting the overproduction of hydroxyl radicals and inducing apoptosis-like death. The widespread use of antibiotics has led to the emergence of drug-resistant bacteria, particularly multidrug-resistant bacteria. MRSA is the most common drug-resistant bacterium that causes suppurative infections in humans. As only a limited number of drugs are available to treat the infections caused by drug-resistant pathogens, it is imperative to develop novel and effective biological agents for treating MRSA infections. This is the first study to show that platelets can inhibit MRSA growth and . Our results revealed that platelets enhanced the production of hydroxyl radicals in MRSA, which induced a series of apoptosis hallmarks in MRSA, including DNA fragmentation, chromosome condensation, phosphatidylserine exposure, membrane potential depolarization, and increased intracellular caspase activity. These findings may further our understanding of platelet function.
Topics: Animals; Anti-Bacterial Agents; Apoptosis; Blood Platelets; Cell Death; Humans; Hydroxyl Radical; Methicillin-Resistant Staphylococcus aureus; Mice; Microbial Sensitivity Tests; Staphylococcal Infections
PubMed: 35852345
DOI: 10.1128/spectrum.02441-21 -
Scientific Reports Oct 2018Complementary vibrational spectroscopic techniques - infrared, Raman and inelastic neutron scattering (INS) - were applied to the study of human bone burned under...
Complementary vibrational spectroscopic techniques - infrared, Raman and inelastic neutron scattering (INS) - were applied to the study of human bone burned under controlled conditions (400 to 1000 °C). This is an innovative way of tackling bone diagenesis upon burning, aiming at a quantitative evaluation of heat-induced dimensional changes allowing a reliable estimation of pre-burning skeletal dimensions. INS results allowed the concomitant observation of the hydroxyl libration (OH), hydroxyl stretching (ν(OH)) and (OH + ν(OH)) combination modes, leading to an unambiguous assignment of these INS features to bioapatite and confirming hydroxylation of bone's inorganic matrix. The OH, ν(OH) and ν(PO) bands were identified as spectral biomarkers, which displayed clear quantitative relationships with temperature revealing heat-induced changes in bone's H-bonding pattern during the burning process. These results will enable the routine use of FTIR-ATR (Fourier Transform Infrared-Attenuated Total Reflectance) for the analysis of burned skeletal remains, which will be of the utmost significance in forensic, bioanthropological and archaeological contexts.
Topics: Biomarkers; Bone and Bones; Hot Temperature; Humans; Hydrogen Bonding; Hydroxyl Radical; Spectrophotometry, Infrared; Spectrum Analysis, Raman
PubMed: 30374054
DOI: 10.1038/s41598-018-34376-w -
Bioinformatics (Oxford, England) Sep 2015A key to understanding RNA function is to uncover its complex 3D structure. Experimental methods used for determining RNA 3D structures are technologically challenging...
UNLABELLED
A key to understanding RNA function is to uncover its complex 3D structure. Experimental methods used for determining RNA 3D structures are technologically challenging and laborious, which makes the development of computational prediction methods of substantial interest. Previously, we developed the iFoldRNA server that allows accurate prediction of short (<50 nt) tertiary RNA structures starting from primary sequences. Here, we present a new version of the iFoldRNA server that permits the prediction of tertiary structure of RNAs as long as a few hundred nucleotides. This substantial increase in the server capacity is achieved by utilization of experimental information such as base-pairing and hydroxyl-radical probing. We demonstrate a significant benefit provided by integration of experimental data and computational methods.
AVAILABILITY AND IMPLEMENTATION
http://ifoldrna.dokhlab.org
CONTACT
Topics: Algorithms; Base Pairing; Computational Biology; Computer Simulation; Hydroxyl Radical; Models, Molecular; Nucleic Acid Conformation; RNA; Sequence Analysis, RNA; Software
PubMed: 25910700
DOI: 10.1093/bioinformatics/btv221 -
Plant, Cell & Environment Aug 2014Plants emit a plethora of volatile organic compounds, which provide detailed information on the physiological condition of emitters. Volatiles induced by herbivore... (Review)
Review
Plants emit a plethora of volatile organic compounds, which provide detailed information on the physiological condition of emitters. Volatiles induced by herbivore feeding are among the best studied plant responses to stress and may constitute an informative message to the surrounding community and further function in plant defence processes. However, under natural conditions, plants are potentially exposed to multiple concurrent stresses with complex effects on the volatile emissions. Atmospheric pollutants are an important facet of the abiotic environment and can impinge on a plant's volatile-mediated defences in multiple ways at multiple temporal scales. They can exert changes in volatile emissions through oxidative stress, as is the case with ozone pollution. The pollutants, in particular, ozone, nitrogen oxides and hydroxyl radicals, also react with volatiles in the atmosphere. These reactions result in volatile breakdown products, which may themselves be perceived by community members as informative signals. In this review, we demonstrate the complex interplay among stresses, emitted signals, and modification in signal strength and composition by the atmosphere, collectively determining the responses of the biotic community to elicited signals.
Topics: Air Pollution; Atmosphere; Hydroxyl Radical; Nitrogen Oxides; Oxidative Stress; Ozone; Plants; Stress, Physiological; Volatile Organic Compounds
PubMed: 24738697
DOI: 10.1111/pce.12352 -
Journal of the American Chemical Society Oct 2022The hydroxyl radical (OH) is one of the most attractive reactive oxygen species due to its high oxidation power and its clean (photo)(electro)generation from water,...
The hydroxyl radical (OH) is one of the most attractive reactive oxygen species due to its high oxidation power and its clean (photo)(electro)generation from water, leaving no residues and creating new prospects for efficient wastewater treatment and electrosynthesis. Unfortunately, in situ detection of OH is challenging due to its short lifetime (few ns). Using lifetime-extending spin traps, such as 5,5-dimethyl-1-pyrroline -oxide (DMPO) to generate the [DMPO-OH] adduct in combination with electron spin resonance (ESR), allows unambiguous determination of its presence in solution. However, this method is cumbersome and lacks the necessary sensitivity and versatility to explore and quantify OH generation dynamics at electrode surfaces in real time. Here, we identify that [DMPO-OH] is redox-active with = 0.85 V vs Ag|AgCl and can be conveniently detected on Au and C ultramicroelectrodes. Using scanning electrochemical microscopy (SECM), a four-electrode technique capable of collecting the freshly generated [DMPO-OH] from near the electrode surface, we detected its generation in real time from operating electrodes. We also generated images of [DMPO-OH] production and estimated and compared its generation efficiency at various electrodes (boron-doped diamond, tin oxide, titanium foil, glassy carbon, platinum, and lead oxide). Density functional calculations, ESR measurements, and bulk calibration using the Fenton reaction helped us unambiguously identify [DMPO-OH] as the source of redox activity. We hope these findings will encourage the rapid, inexpensive, and quantitative detection of OH for conducting informed explorations of its role in mediated oxidation processes at electrode surfaces for energy, environmental, and synthetic applications.
Topics: Hydroxyl Radical; Reactive Oxygen Species; Microscopy, Electrochemical, Scanning; Platinum; Titanium; Boron; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Oxidation-Reduction; Electrodes; Water; Carbon; Diamond; Free Radicals; Spin Labels
PubMed: 36215201
DOI: 10.1021/jacs.2c06278 -
Chemical Society Reviews Sep 2020Contrary to frequent reports in the literature, hydroxyl radical is not a key species participating in endogenous oxidative DNA damage. Instead, carbonate radical anion... (Review)
Review
Contrary to frequent reports in the literature, hydroxyl radical is not a key species participating in endogenous oxidative DNA damage. Instead, carbonate radical anion is formed from the Fenton reaction under cellular conditions and from decomposition of nitrosoperoxycarbonate generated during inflammation. Carbonate radical anion is a potent one-electron oxidant capable of generating base radical cations that can migrate over long distances in duplex DNA, ultimately generating 8-oxo-7,8-dihydroguanine at a redox-sensitive sequence such as GGG. Such a mechanism enables G-quadruplex-forming sequences to act as long-range sensors of oxidative stress, impacting gene expression via the DNA repair mechanism that reads and ultimately erases the oxidized base. With a writing, reading and erasing mechanism in place, oxidative 'damage' to DNA might be relabeled as 'epigenetic' modifications.
Topics: DNA; DNA Damage; Epigenomics; Hydroxyl Radical; Oxidative Stress
PubMed: 32785348
DOI: 10.1039/d0cs00579g -
Analytical Chemistry Dec 2023Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a...
Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.
Topics: Edetic Acid; Hydroxyl Radical; Proteins; Protein Footprinting; Oxidative Stress; Oxidation-Reduction
PubMed: 38049117
DOI: 10.1021/acs.analchem.3c02319 -
Biochemistry Feb 2010Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters... (Review)
Review
Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of "ordered" waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.
Topics: Animals; Crystallography, X-Ray; Humans; Hydroxyl Radical; Magnetic Resonance Spectroscopy; Membrane Proteins; Protein Footprinting; Receptors, G-Protein-Coupled; Rhodopsin; Water
PubMed: 20047303
DOI: 10.1021/bi901889t