-
Journal of Visualized Experiments : JoVE Jun 2021Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein...
Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (OH) to irreversibly label a protein's solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.
Topics: Hydroxyl Radical; Lasers; Oxidation-Reduction; Protein Footprinting; Proteins; Reproducibility of Results
PubMed: 34152327
DOI: 10.3791/61861 -
Archives of Toxicology Jul 2022The toxic potential of HO is limited, even if intracellular concentrations of HO under conditions of oxidative stress increase to the micromolar concentration range. Its... (Review)
Review
The toxic potential of HO is limited, even if intracellular concentrations of HO under conditions of oxidative stress increase to the micromolar concentration range. Its toxicity is mostly restricted to the oxidation of highly reactive thiol groups, some of which are functionally very important. Subsequently, the HO radical is generated spontaneously from HO in the Fenton reaction. The HO radical is extremely toxic and destroys any biological structure. Due to the high reactivity, its action is limited to a locally restricted site of its generation. On the other hand, HO with its stability and long half-life can reach virtually any site and distribute its toxic effect all over the cell. Thereby HO, in spite of its ultra-short half-life (10 s), can execute its extraordinary toxic action at any target of the cell. In this oxidative stress scenario, HO is the pro-radical, that spreads the toxic action of the HO radical. It is the longevity of the HO molecule allowing it to distribute its toxic action from the site of origin all over the cell and may even mediate intercellular communication. Thus, HO acts as a spreader by transporting it to sites where the extremely short-lived toxic HO radical can arise in the presence of "free iron". HO and HO act in concert due to their different complementary chemical properties. They are dependent upon each other while executing the toxic effects in oxidative stress under diabetic metabolic conditions in particular in the highly vulnerable pancreatic beta cell, which in contrast to many other cell types is so badly protected against oxidative stress due to its extremely low HO inactivating enzyme capacity.
Topics: Hydrogen Peroxide; Hydroxyl Radical; Insulin-Secreting Cells; Iron; Oxidation-Reduction
PubMed: 35416515
DOI: 10.1007/s00204-022-03282-6 -
Oxidative Medicine and Cellular... 2011It is generally believed that diseases caused by oxidative stress should be treated with antioxidants. However, clinical trials with such antioxidants as ascorbic acid... (Review)
Review
It is generally believed that diseases caused by oxidative stress should be treated with antioxidants. However, clinical trials with such antioxidants as ascorbic acid and vitamin E, failed to produce the expected beneficial results. On the other hand, important biomolecules can be modified by the introduction of oxygen atoms by means of non-oxidative hydroxyl radicals. In addition, hydroxyl radicals can reduce disulfide bonds in proteins, specifically fibrinogen, resulting in their unfolding and scrambled refolding into abnormal spatial configurations. Consequences of this reaction are observed in many diseases such as atherosclerosis, cancer and neurological disorders, and can be prevented by the action of non-reducing substances. Moreover, many therapeutic substances, traditionally classified as antioxidants, accept electrons and thus are effective oxidants. It is described in this paper that hydroxyl radicals can be generated by ferric ions without any oxidizing agent. In view of the well-known damaging effect of poorly chelated iron in the human body, numerous natural products containing iron binding agents can be essential in the maintenance of human health. However, beneficial effects of the great number of phytochemicals that are endowed with hydroxyl radical scavenging and/or iron chelating activities should not be considered as a proof for oxidative stress.
Topics: Animals; Free Radical Scavengers; Humans; Hydroxyl Radical; Models, Biological; Oxidative Stress
PubMed: 21904647
DOI: 10.1155/2011/809696 -
Protein and Peptide Letters 2019Determination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry... (Review)
Review
BACKGROUND
Determination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry (MS). One approach is hydroxyl radical protein footprinting whereby amino-acid side chains are modified with reactive reagents to modify irreversibly a protein side chain. The outcomes, when deciphered with mass-spectrometry-based proteomics, can increase our knowledge of structure, assembly, and conformational dynamics of macromolecules in solution. Generating the hydroxyl radicals by laser irradiation, Hambly and Gross developed the approach of Fast Photochemical Oxidation of Proteins (FPOP), which labels proteins on the sub millisecond time scale and provides, with MS analysis, deeper understanding of protein structure and protein-ligand and protein- protein interactions. This review highlights the fundamentals of FPOP and provides descriptions of hydroxyl-radical and other radical and carbene generation, of the hydroxyl labeling of proteins, and of determination of protein modification sites. We also summarize some recent applications of FPOP coupled with MS in protein footprinting.
CONCLUSION
We survey results that show the capability of FPOP for qualitatively measuring protein solvent accessibility on the residue level. To make these approaches more valuable, we describe recent method developments that increase FPOP's quantitative capacity and increase the spatial protein sequence coverage. To improve FPOP further, several new labeling reagents including carbenes and other radicals have been developed. These growing improvements will allow oxidative- footprinting methods coupled with MS to play an increasingly significant role in determining the structure and dynamics of macromolecules and their assemblies.
Topics: Amyloid; Epitope Mapping; Hydroxyl Radical; Mass Spectrometry; Oxidation-Reduction; Photochemical Processes; Protein Folding; Protein Footprinting; Proteins
PubMed: 30484399
DOI: 10.2174/0929866526666181128124554 -
IUBMB Life Jun 2017Iron is an essential element for almost all organisms on Earth. It is necessary for a number of crucial processes such as hemoglobin and myoglobin transport and storage... (Review)
Review
Iron is an essential element for almost all organisms on Earth. It is necessary for a number of crucial processes such as hemoglobin and myoglobin transport and storage of oxygen in mammals; electron transfer support in a variety of iron-sulfur protein or cytochrome reactions; and activation and catalysis of reactions of a wide range of substrate like alkanes, olefins, and alcohols. Living organisms adopted iron as the main metal to carry out all of these functions due to the rich coordination chemistry of its two main redox states, Fe and Fe , and because of its abundance in the Earth's crust and oceans. This paper presents an overview of the coordination chemistry of iron that makes it suitable for a large variety of functions within biological systems. Despite iron's chemical advantages, organisms were forced to manage with some drawbacks: Fe insolubility and the formation of toxic radicals, especially the hydroxyl radical. Iron chemistry within biology is an example of how organisms evolved by creating molecular machinery to overcome these difficulties and perform crucial processes with extraordinary elegance and efficiency. © 2017 IUBMB Life, 69(6):382-388, 2017.
Topics: Biological Transport; Coordination Complexes; Eukaryota; Hemoglobins; Hydroxyl Radical; Iron; Iron-Sulfur Proteins; Myoglobin; Oxidation-Reduction; Oxygen; Prokaryotic Cells
PubMed: 28150902
DOI: 10.1002/iub.1602 -
Biochimica Et Biophysica Acta. Proteins... Sep 2022Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical footprinting approach whereby radicals, produced by UV laser photolysis of hydrogen peroxide,... (Review)
Review
Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical footprinting approach whereby radicals, produced by UV laser photolysis of hydrogen peroxide, induce oxidation of amino acid side-chains. Mass Spectrometry (MS) is employed to locate and quantify the resulting irreversible, covalent oxidations to use as a surrogate for side-chain solvent accessibility. Modulation of oxidation levels under different conditions allows for the characterisation of protein conformation, dynamics and binding epitopes. FPOP has been applied to structurally diverse and biopharmaceutically relevant systems from small, monomeric aggregation-prone proteins to proteome-wide analysis of whole organisms. This review evaluates the current state of FPOP, the progress needed to address data analysis bottlenecks, particularly for residue-level analysis, and highlights significant developments of the FPOP platform that have enabled its versatility and complementarity to other structural biology techniques.
Topics: Hydroxyl Radical; Mass Spectrometry; Oxidation-Reduction; Protein Conformation; Proteins
PubMed: 35933084
DOI: 10.1016/j.bbapap.2022.140829 -
Poultry Science Feb 2019As the most abundant protein in chicken eggs, ovalbumin plays an important role in the processing of high value-added poultry products. The present study investigated...
As the most abundant protein in chicken eggs, ovalbumin plays an important role in the processing of high value-added poultry products. The present study investigated the effects of hydroxyl radical-induced early stage oxidation on the physicochemical and interfacial properties of chicken egg white ovalbumin. Protein carbonyl content of ovalbumin increased (from 0.78 to 1.13 nmol/mg) with the oxidation time (0-5 h), while free sulfhydryl content (from 0.43 to 0.09 nmol/mg) and free amino group content (from 0.49 to 0.43 nmol/mg) decreased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the exposure of ovalbumin to hydroxyl radicals caused self-cross-linking and resulted in the formation of dimers and trimers. Accompanied by these changes, the surface hydrophobicity of ovalbumin was enhanced about 1.5-fold with the deepening of oxidation, and the value of zeta potential became more negative (from -7.15 to -20.51 mv). About 2 h of moderate oxidation improved the foaming and emulsifying properties of ovalbumin (1.2-fold to 1.8-fold), while excessive oxidation (3 h) decreased these interface properties. Hydroxyl radical-induced oxidation changed the surface chemical groups and structures of ovalbumin, thereby affecting the surface properties. The foaming and emulsifying properties of ovalbumin could be improved by oxidation, increasing the application possibilities of ovalbumin in the food interface system.
Topics: Animals; Avian Proteins; Chickens; Egg White; Electrophoresis, Polyacrylamide Gel; Emulsions; Hydroxyl Radical; Ovalbumin; Oxidation-Reduction; Protein Carbonylation; Surface Properties
PubMed: 30101285
DOI: 10.3382/ps/pey370 -
Chemical Reviews Apr 2022Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry has been successfully used to investigate a plethora of protein-related questions. The method,... (Review)
Review
Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry has been successfully used to investigate a plethora of protein-related questions. The method, which utilizes hydroxyl radicals to oxidatively modify solvent-accessible amino acids, can inform on protein interaction sites and regions of conformational change. Hydroxyl radical-based footprinting was originally developed to study nucleic acids, but coupling the method with mass spectrometry has enabled the study of proteins. The method has undergone several advancements since its inception that have increased its utility for more varied applications such as protein folding and the study of biotherapeutics. In addition, recent innovations have led to the study of increasingly complex systems including cell lysates and intact cells. Technological advances have also increased throughput and allowed for better control of experimental conditions. In this review, we provide a brief history of the field of HRPF and detail recent innovations and applications in the field.
Topics: Hydroxyl Radical; Mass Spectrometry; Protein Folding; Protein Footprinting; Proteins
PubMed: 34633178
DOI: 10.1021/acs.chemrev.1c00432 -
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 -
Annual Review of Physical Chemistry Apr 2022Knowledge of protein structure is crucial to our understanding of biological function and is routinely used in drug discovery. High-resolution techniques to determine... (Review)
Review
Knowledge of protein structure is crucial to our understanding of biological function and is routinely used in drug discovery. High-resolution techniques to determine the three-dimensional atomic coordinates of proteins are available. However, such methods are frequently limited by experimental challenges such as sample quantity, target size, and efficiency. Structural mass spectrometry (MS) is a technique in which structural features of proteins are elucidated quickly and relatively easily. Computational techniques that convert sparse MS data into protein models that demonstrate agreement with the data are needed. This review features cutting-edge computational methods that predict protein structure from MS data such as chemical cross-linking, hydrogen-deuterium exchange, hydroxyl radical protein footprinting, limited proteolysis, ion mobility, and surface-induced dissociation. Additionally, we address future directions for protein structure prediction with sparse MS data.
Topics: Hydroxyl Radical; Mass Spectrometry; Protein Conformation; Protein Footprinting; Proteins
PubMed: 34724394
DOI: 10.1146/annurev-physchem-082720-123928