This study examines the redox sensitivity of a conserved residue in myosin II that regulates muscle contraction. Specifically:
1) A methionine residue (M394) in Dictyostelium myosin II that regulates ATPase activity is conserved as a cysteine in human cardiac and skeletal myosins.
2) Oxidation of M394 methionine or mutation to glutamine reduced ATPase activity, while mutation to leucine prevented effects of oxidation. Reduction of oxidized M394 by methionine sulfoxide reductase restored activity.
3) Mutation of M394 to cysteine allowed reversible inhibition of ATPase activity through glutathionylation and reduction, mimicking effects
This document summarizes the synthesis of novel 3'-C-methylene thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates and phosphonamidites for modifying the backbone of oligonucleotides. The key intermediates 3'-C-iodomethyl nucleosides were reacted with in situ generated bis(trimethylsilyl)phosphite (BTSP) using modified Arbuzov reaction conditions to directly obtain the desired 5'-O-DMT and MMT protected 3'-C-methylene modified H-phosphonates without removing protecting groups. Some phosphonates were further converted to corresponding phosphonamidite monomers through a one-pot multi
This document summarizes a study that used X-ray crystallography and resonance Raman spectroscopy to characterize the oxo-molybdenum dithiolene complex (Tp*)MoO(qdt), which is a model system relevant to pyranopterin molybdenum enzyme active sites like sulfite oxidase. The compound was found to crystallize in the triclinic space group P1. Structural parameters of the first coordination sphere were similar to the related complex (Tp*)MoO(bdt), suggesting geometric effects are not major contributors to electronic structural differences between the two complexes. Raman spectroscopy provided evidence the dithiolene ligand interacts with the molybdenum center through
Characterization of mg state of mb in presence of peg 10 (z a parray) originalZahoor Parray
This document summarizes a study that characterized the intermediate state of myoglobin (Mb) in the presence of polyethylene glycol 10 (PEG 10) under physiological conditions. The researchers found that PEG 10 perturbed the tertiary structure of Mb but did not significantly change its secondary structure. PEG 10 was found to induce a molten globule state in Mb, where the intermediate state had hydrophobic patches and a larger hydrodynamic volume than the native protein. Isothermal titration calorimetry showed strong binding between Mb and PEG 10 at physiological pH. The researchers hypothesize that PEG 10 induces a molten globule conformation in Mb by interacting with its heme group. They conclude that protein-crowder interactions need careful
This study investigated the structural, thermodynamic and unfolding properties of the kappa class glutathione transferase (CdGSTK1-1) from Camelus dromedarius. The key findings were:
1) CdGSTK1-1 was expressed in E. coli and purified. Analytical gel filtration showed it has a unique trimeric structure.
2) CdGSTK1-1 exhibited low thermal stability and unfolded through three states with intermediate species formation. The first transition melting point was 40.3°C and the second was 49.1°C.
3) Intrinsic fluorescence and near-UV CD studies indicated that substrate binding did not cause major conformational changes in
Metal cu(ii) and zn(ii) bipyridyls as inhibitors of lactaterkkoiri
This document describes a study examining how metal complexes of copper (Cu-bpy) and zinc (Zn-bpy) interact with and modulate the activity of lactate dehydrogenase (LDH) in mouse tissues. The key findings are:
1) Both Cu-bpy and Zn-bpy were found to bind to LDH, with Zn-bpy showing a stronger binding constant.
2) Treatment of mouse tissues with non-toxic doses of the complexes resulted in a significant decline in LDH activity in various tissues, with Cu-bpy inhibiting LDH in all tissues studied and Zn-bpy inhibiting LDH in liver, kidney, and heart tissues.
3) Electrophoresis analysis suggested that both complexes equally inhibited
This study investigated the effects of spermine NONOate (a nitric oxide donor) and ATP on the thermal aggregation of albumin and hemoglobin proteins using light scattering. The main findings were:
1) Spermine NONOate decreased the temperature at which hemoglobin aggregates, regardless of sodium/potassium environment.
2) ATP alone did not affect protein thermal stability but facilitated protein destabilization in the presence of spermine NONOate.
3) The effects of ATP and nitric oxide were strongly influenced by the buffer's ionic composition of sodium versus potassium.
In-vitro biological activities of the free new H4L ( indole-7-thiocarbohydrazone) ligand and its Ni(II), Pd(II) , Pt(II),
Cu(II), Ag(I), Zn(II) and Cd(II) complexes are screened against two cancerous cell lines, that revealed significant
activity only for [Cu2Cl2(H4L)2(PPh3)2] after 72 h treatment by the highest tested concentrations. The Copper(I)
complex was characterized by X-ray Crystallography and the NMR spectra, whereas it has been confirmed to have
momentous cytotoxicity against ovarian, breast cancerous cell lines (Caov-3, MCF-7). The apoptosis-inducing
properties of the Cu(I) complex have been investigated through fluorescence microscopy visualization, DNA
fragmentation analysis and propidium iodide flow cytometry.
The document describes a thesis submitted by TeQuion Brookins on March 26, 2012 about the influence of the protein cross-linker diethyl acetylenedicarboxylate (DAD) on degradation of proteins by the 20S core particle of the 26S proteasome. The thesis explores how DAD-mediated electrophilic modification can cross-link proteins like the peroxiredoxin Tsa1, inactivate them, and potentially inhibit their degradation by the proteasome. Experiments were conducted to detect increased accumulation of ubiquitin, known proteasomal substrates, and damaged proteins like Tsa1 when yeast cells were treated with DAD, which would indicate disturbance of the cellular protein degradation mechanism. P
This document summarizes the synthesis of novel 3'-C-methylene thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates and phosphonamidites for modifying the backbone of oligonucleotides. The key intermediates 3'-C-iodomethyl nucleosides were reacted with in situ generated bis(trimethylsilyl)phosphite (BTSP) using modified Arbuzov reaction conditions to directly obtain the desired 5'-O-DMT and MMT protected 3'-C-methylene modified H-phosphonates without removing protecting groups. Some phosphonates were further converted to corresponding phosphonamidite monomers through a one-pot multi
This document summarizes a study that used X-ray crystallography and resonance Raman spectroscopy to characterize the oxo-molybdenum dithiolene complex (Tp*)MoO(qdt), which is a model system relevant to pyranopterin molybdenum enzyme active sites like sulfite oxidase. The compound was found to crystallize in the triclinic space group P1. Structural parameters of the first coordination sphere were similar to the related complex (Tp*)MoO(bdt), suggesting geometric effects are not major contributors to electronic structural differences between the two complexes. Raman spectroscopy provided evidence the dithiolene ligand interacts with the molybdenum center through
Characterization of mg state of mb in presence of peg 10 (z a parray) originalZahoor Parray
This document summarizes a study that characterized the intermediate state of myoglobin (Mb) in the presence of polyethylene glycol 10 (PEG 10) under physiological conditions. The researchers found that PEG 10 perturbed the tertiary structure of Mb but did not significantly change its secondary structure. PEG 10 was found to induce a molten globule state in Mb, where the intermediate state had hydrophobic patches and a larger hydrodynamic volume than the native protein. Isothermal titration calorimetry showed strong binding between Mb and PEG 10 at physiological pH. The researchers hypothesize that PEG 10 induces a molten globule conformation in Mb by interacting with its heme group. They conclude that protein-crowder interactions need careful
This study investigated the structural, thermodynamic and unfolding properties of the kappa class glutathione transferase (CdGSTK1-1) from Camelus dromedarius. The key findings were:
1) CdGSTK1-1 was expressed in E. coli and purified. Analytical gel filtration showed it has a unique trimeric structure.
2) CdGSTK1-1 exhibited low thermal stability and unfolded through three states with intermediate species formation. The first transition melting point was 40.3°C and the second was 49.1°C.
3) Intrinsic fluorescence and near-UV CD studies indicated that substrate binding did not cause major conformational changes in
Metal cu(ii) and zn(ii) bipyridyls as inhibitors of lactaterkkoiri
This document describes a study examining how metal complexes of copper (Cu-bpy) and zinc (Zn-bpy) interact with and modulate the activity of lactate dehydrogenase (LDH) in mouse tissues. The key findings are:
1) Both Cu-bpy and Zn-bpy were found to bind to LDH, with Zn-bpy showing a stronger binding constant.
2) Treatment of mouse tissues with non-toxic doses of the complexes resulted in a significant decline in LDH activity in various tissues, with Cu-bpy inhibiting LDH in all tissues studied and Zn-bpy inhibiting LDH in liver, kidney, and heart tissues.
3) Electrophoresis analysis suggested that both complexes equally inhibited
This study investigated the effects of spermine NONOate (a nitric oxide donor) and ATP on the thermal aggregation of albumin and hemoglobin proteins using light scattering. The main findings were:
1) Spermine NONOate decreased the temperature at which hemoglobin aggregates, regardless of sodium/potassium environment.
2) ATP alone did not affect protein thermal stability but facilitated protein destabilization in the presence of spermine NONOate.
3) The effects of ATP and nitric oxide were strongly influenced by the buffer's ionic composition of sodium versus potassium.
In-vitro biological activities of the free new H4L ( indole-7-thiocarbohydrazone) ligand and its Ni(II), Pd(II) , Pt(II),
Cu(II), Ag(I), Zn(II) and Cd(II) complexes are screened against two cancerous cell lines, that revealed significant
activity only for [Cu2Cl2(H4L)2(PPh3)2] after 72 h treatment by the highest tested concentrations. The Copper(I)
complex was characterized by X-ray Crystallography and the NMR spectra, whereas it has been confirmed to have
momentous cytotoxicity against ovarian, breast cancerous cell lines (Caov-3, MCF-7). The apoptosis-inducing
properties of the Cu(I) complex have been investigated through fluorescence microscopy visualization, DNA
fragmentation analysis and propidium iodide flow cytometry.
The document describes a thesis submitted by TeQuion Brookins on March 26, 2012 about the influence of the protein cross-linker diethyl acetylenedicarboxylate (DAD) on degradation of proteins by the 20S core particle of the 26S proteasome. The thesis explores how DAD-mediated electrophilic modification can cross-link proteins like the peroxiredoxin Tsa1, inactivate them, and potentially inhibit their degradation by the proteasome. Experiments were conducted to detect increased accumulation of ubiquitin, known proteasomal substrates, and damaged proteins like Tsa1 when yeast cells were treated with DAD, which would indicate disturbance of the cellular protein degradation mechanism. P
1) The document comments on a previous study that found microbial oxidation of cobalt and manganese in seawater samples is correlated and may occur via a common enzymatic pathway.
2) The comment proposes that wood-degrading fungi and their extracellular enzyme manganese peroxidase (MnP) provide an alternative explanation for oxidation of cobalt, manganese, and cerium in seawater and their transition from dissolved to particulate phases.
3) MnP is known to oxidize manganese and cobalt, and likely also oxidizes cerium, supporting the idea that these three metals may be oxidized via a common enzymatic pathway involving MnP.
This document contains course outlines for several biology courses, including Developmental Biology, Cell & Molecular Biology, Animal Behaviour, Biochemistry, and Ecology. The Developmental Biology course covers topics like spermatogenesis, fertilization, cleavage, gastrulation, early vertebrate development, and organogenesis. The Cell & Molecular Biology course focuses on cellular structures like the plasma membrane, cytoskeleton, and organelles, as well as processes like DNA replication, transcription, and translation. The Animal Behaviour course examines the mechanisms of behavior and social organization. The Biochemistry course covers amino acids, enzymes, carbohydrates, lipids, vitamins, metabolism, and nitrogen metabolism. Finally, the Ecology course outlines concepts in
Glycosylation is an important post-translational modification that affects protein structure, stability, and function in various ways. It involves attaching carbohydrate chains to proteins. There are two main types of glycosylation - N-linked and O-linked. Glycosylation helps in cell recognition and adhesion, can modulate enzyme activity, improves protein solubility and stability, and is involved in receptor signaling. It can adjust the thermodynamic, kinetic and structural properties of proteins. Glycosylation increases protein stability by destabilizing the unfolded state more than it stabilizes the folded state.
1) The document describes a novel method called INLIGHTTM for the relative quantification of N-linked glycans using isotopically labeled glycan hydrazide tags.
2) The method involves releasing N-linked glycans from glycoproteins using PNGase F, then derivatizing the glycans from two samples with either a light or heavy tagged reagent.
3) The tagged glycans are mixed, analyzed by LC/MS, and ratios of light and heavy glycan pairs are calculated to quantify differences between the two samples after correcting for isotopic overlap.
This document appears to be notes from lectures given by Dr. Ashok Kumar Jeppu on the topics of enzyme inhibition and allosteric regulation. It discusses various types of enzyme inhibition including competitive, non-competitive and irreversible inhibition. It provides examples of drugs that work through different inhibition mechanisms like DFMO and aspirin. The document also covers the concept of allosteric effectors and how binding of an allosteric effector can induce a conformational change in the enzyme to increase or decrease its activity. It gives aspartate transcarbamoylase as an example of an enzyme subjected to feedback allosteric inhibition.
The document discusses ruthenium-based drugs as potential alternatives to platinum-based anticancer drugs like cisplatin. Ruthenium compounds have properties that make them well-suited for medical applications, including ligand exchange kinetics similar to cisplatin and the ability to exist in multiple oxidation states under physiological conditions. Some ruthenium complexes have shown anticancer activity through interactions with DNA like intercalation and groove binding. The most successful ruthenium-based drug to date is NAMI-A, which can bind DNA but does not appear to cause DNA damage as its mechanism of action.
This document summarizes the synthesis and characterization of the oxomolybdenum mono-ene-1,2-dithiolate complex (Tp*)MoO(bdtCl2) (3). X-ray crystallography showed compound 3 crystallizes in the monoclinic space group P21/c, with a distorted octahedral coordination geometry around the Mo atom. IR, EPR, and electronic absorption spectroscopy indicate the chlorine substituents do not significantly perturb the electronic structure of the Mo(V) center, but solution redox potentials and gas-phase ionization energies are sensitive to remote substituent effects. The coordination environment of 3 is similar to the related complex (Tp*)MoO(
2012_Nature_Synthetic Homeostatic MaterialsXimin He
This document describes a new materials platform called SMARTS (self-regulated mechanochemical adaptively reconfigurable tunable system) that can create self-regulating homeostatic materials. The system uses a hydrogel embedded with microstructures that reversibly actuate between upright and bent positions in response to a chemical stimulus. This mechanical actuation turns chemical reactions on and off by moving a catalyst in and out of a reactant layer. Various examples are given that demonstrate precise triggering of chemical reactions in synchronization with the driving stimulus. The system can also be designed to maintain homeostatic feedback loops where a chemical output regulates the initial stimulus, effectively maintaining a stable parameter like temperature.
This document summarizes a research project characterizing a novel DMSP-producing bacterium, Novosphingobium sp. MBES04. The introduction provides background on DMSP, its global production, roles in marine ecosystems, and known biosynthesis pathways. The student isolated DMSP-producing bacteria from a saltmarsh, identifying Novosphingobium sp. MBES04. Experiments showed for the first time that this alphaproteobacterium produces DMSP through the methylation pathway used by plants, not the pathway previously identified in bacteria. Characterization demonstrated DMSP likely serves as an osmoprotectant and is produced more under low nitrogen conditions.
This document outlines a lecture on carbohydrate and nucleic acid chemistry. It begins by introducing carbohydrates as one of the four major classes of biological molecules, along with proteins, lipids, and nucleic acids. Carbohydrates serve important nutritional, structural, informational, and regulatory functions in living systems. They are classified based on their monomer units into monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The lecture further discusses carbohydrate isomers, epimers, enantiomers, cyclization, and polysaccharides such as starch, glycogen, cellulose, and chitin. It then introduces nucleic acids DNA and RNA as polymers of nucleotides, highlighting their monomers, base pairing, and DNA
Metabolism is the sum of all chemical reactions within an organism. Metabolic pathways consist of enzyme-catalyzed reactions organized into linear chains or cyclic pathways. Enzymes lower the activation energy of reactions by binding to substrates and altering their structure. Enzyme inhibitors can competitively or non-competitively bind to enzymes and reduce their activity. Metabolic pathways are regulated by end-product inhibition, which stops pathways once their product reaches a certain concentration. Databases can be used to screen chemicals and identify potential new drugs, like anti-malarial treatments. Rates of enzymatic reactions can be calculated from experimental data and plotted on graphs to determine inhibition type.
1. The study characterized the aggregation of recombinant human Interleukin-1 receptor type II (rhuIL-1RII) using differential scanning calorimetry (DSC) and size exclusion chromatography (SEC).
2. A scan-rate dependence in the DSC experiment and a break from linearity in initial aggregation rates near the melting temperature (Tm) suggested that protein unfolding significantly contributes to the aggregation reaction pathway.
3. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from the irreversibly denatured aggregation process by simulating how unfolding properties could predict aggregation rates at different temperatures above and below the Tm.
This document describes research on using genetically fused gold-binding peptides (GBP1) to direct the self-immobilization of alkaline phosphatase (AP) enzymes on micro-patterned gold substrates. GBP1 was genetically fused to the N-terminus of AP in tandem repeats of 5, 6, 7, and 9 units (nGBP1-AP). The 5GBP1-AP fusion construct displayed the best binding and enzymatic activity. Surface plasmon resonance spectroscopy and atomic force microscopy showed that 5GBP1-AP self-assembled and immobilized on gold surfaces. Immobilized 5GBP1-AP exhibited higher enzymatic activity per area than unmodified AP. This demonstrates the potential
This document describes the total synthesis of bleomycin A2 and related compounds. Two total syntheses of bleomycin A2 are reported that use bleomycin demethyl A2 as a key intermediate. A synthesis of decarbamoyl bleomycin demethyl A2 is also described, which represents the first synthetic access to this bleomycin congener. Improved methods for synthesizing the carbohydrate moiety of bleomycin are explored. Synthetic bleomycin was shown to have the same biological activity and DNA cleavage selectivity as naturally occurring bleomycin.
This document summarizes research on the reduction of manganese porphyrins by flavoenzymes and mitochondrial particles, and how this relates to the catalytic reduction of peroxynitrite. It finds that flavoenzymes like xanthine oxidase and glucose oxidase, as well as mitochondrial Complex I and II, catalyze the reduction of manganese porphyrins using substrates like NADH and succinate. Reduced manganese porphyrins can then rapidly react with peroxynitrite via a two-electron process, producing nitrite and manganese porphyrin radicals. This redox cycle can protect peroxynitrite-sensitive targets in submitochondrial particles.
This document describes a new method for selective N-methylation of peptide backbone amides on resin using the Mitsunobu reaction. The key aspects are:
1) N-trifluoroacetamide (Tfa) was used as the protecting group on resin-bound peptides, which can generate a nucleophilic anion for methylation via Mitsunobu conditions.
2) Tfa-protected peptides on resin underwent efficient (80-99%) and selective N-methylation of the backbone amide using the Mitsunobu reaction with triphenylphosphine, methanol and diisopropyl azodicarboxylate.
3) Unlike other reports, the T
This document discusses enzymes and how they control metabolism in cells. It provides details on how enzymes have an active site that binds to specific substrates, and how enzyme catalysis involves molecular motion and substrate collision with the active site. It describes how temperature, pH, and substrate concentration can affect enzyme activity, and includes graphs to illustrate these relationships. The document discusses enzyme inhibition, denaturation of enzymes, and industrial uses of immobilized enzymes. It also covers the production of lactose-free milk and provides guidance on designing an experiment to test the effects of various factors on enzyme activity.
1) The olsA gene mediates the synthesis of ornithine lipids in P. aeruginosa under phosphate-limiting conditions. Ornithine lipids are phosphate-free and their production allows the bacteria to reduce phosphate utilization in its membranes.
2) Mass spectrometry analysis confirmed the production and structure of ornithine lipids in P. aeruginosa, which contain mainly C16:0 and C18:1 fatty acid chains.
3) While resistance to antimicrobial peptides increases under phosphate limitation, ornithine lipid production was found to not be required for this increased resistance or for virulence in a C. elegans infection model.
1. El documento presenta la agenda del primer día de una asignatura. Incluye la bienvenida a los estudiantes, la presentación del profesor y los objetivos del curso. También cubre las expectativas de los alumnos, el programa, el instructivo académico y una sesión de preguntas y respuestas.
2. La unidad uno trata sobre conceptos básicos de didáctica como aprendizaje, currículo, enseñanza y educación. Explica los tipos de didáctica y los paradigmas educativos.
1) The document comments on a previous study that found microbial oxidation of cobalt and manganese in seawater samples is correlated and may occur via a common enzymatic pathway.
2) The comment proposes that wood-degrading fungi and their extracellular enzyme manganese peroxidase (MnP) provide an alternative explanation for oxidation of cobalt, manganese, and cerium in seawater and their transition from dissolved to particulate phases.
3) MnP is known to oxidize manganese and cobalt, and likely also oxidizes cerium, supporting the idea that these three metals may be oxidized via a common enzymatic pathway involving MnP.
This document contains course outlines for several biology courses, including Developmental Biology, Cell & Molecular Biology, Animal Behaviour, Biochemistry, and Ecology. The Developmental Biology course covers topics like spermatogenesis, fertilization, cleavage, gastrulation, early vertebrate development, and organogenesis. The Cell & Molecular Biology course focuses on cellular structures like the plasma membrane, cytoskeleton, and organelles, as well as processes like DNA replication, transcription, and translation. The Animal Behaviour course examines the mechanisms of behavior and social organization. The Biochemistry course covers amino acids, enzymes, carbohydrates, lipids, vitamins, metabolism, and nitrogen metabolism. Finally, the Ecology course outlines concepts in
Glycosylation is an important post-translational modification that affects protein structure, stability, and function in various ways. It involves attaching carbohydrate chains to proteins. There are two main types of glycosylation - N-linked and O-linked. Glycosylation helps in cell recognition and adhesion, can modulate enzyme activity, improves protein solubility and stability, and is involved in receptor signaling. It can adjust the thermodynamic, kinetic and structural properties of proteins. Glycosylation increases protein stability by destabilizing the unfolded state more than it stabilizes the folded state.
1) The document describes a novel method called INLIGHTTM for the relative quantification of N-linked glycans using isotopically labeled glycan hydrazide tags.
2) The method involves releasing N-linked glycans from glycoproteins using PNGase F, then derivatizing the glycans from two samples with either a light or heavy tagged reagent.
3) The tagged glycans are mixed, analyzed by LC/MS, and ratios of light and heavy glycan pairs are calculated to quantify differences between the two samples after correcting for isotopic overlap.
This document appears to be notes from lectures given by Dr. Ashok Kumar Jeppu on the topics of enzyme inhibition and allosteric regulation. It discusses various types of enzyme inhibition including competitive, non-competitive and irreversible inhibition. It provides examples of drugs that work through different inhibition mechanisms like DFMO and aspirin. The document also covers the concept of allosteric effectors and how binding of an allosteric effector can induce a conformational change in the enzyme to increase or decrease its activity. It gives aspartate transcarbamoylase as an example of an enzyme subjected to feedback allosteric inhibition.
The document discusses ruthenium-based drugs as potential alternatives to platinum-based anticancer drugs like cisplatin. Ruthenium compounds have properties that make them well-suited for medical applications, including ligand exchange kinetics similar to cisplatin and the ability to exist in multiple oxidation states under physiological conditions. Some ruthenium complexes have shown anticancer activity through interactions with DNA like intercalation and groove binding. The most successful ruthenium-based drug to date is NAMI-A, which can bind DNA but does not appear to cause DNA damage as its mechanism of action.
This document summarizes the synthesis and characterization of the oxomolybdenum mono-ene-1,2-dithiolate complex (Tp*)MoO(bdtCl2) (3). X-ray crystallography showed compound 3 crystallizes in the monoclinic space group P21/c, with a distorted octahedral coordination geometry around the Mo atom. IR, EPR, and electronic absorption spectroscopy indicate the chlorine substituents do not significantly perturb the electronic structure of the Mo(V) center, but solution redox potentials and gas-phase ionization energies are sensitive to remote substituent effects. The coordination environment of 3 is similar to the related complex (Tp*)MoO(
2012_Nature_Synthetic Homeostatic MaterialsXimin He
This document describes a new materials platform called SMARTS (self-regulated mechanochemical adaptively reconfigurable tunable system) that can create self-regulating homeostatic materials. The system uses a hydrogel embedded with microstructures that reversibly actuate between upright and bent positions in response to a chemical stimulus. This mechanical actuation turns chemical reactions on and off by moving a catalyst in and out of a reactant layer. Various examples are given that demonstrate precise triggering of chemical reactions in synchronization with the driving stimulus. The system can also be designed to maintain homeostatic feedback loops where a chemical output regulates the initial stimulus, effectively maintaining a stable parameter like temperature.
This document summarizes a research project characterizing a novel DMSP-producing bacterium, Novosphingobium sp. MBES04. The introduction provides background on DMSP, its global production, roles in marine ecosystems, and known biosynthesis pathways. The student isolated DMSP-producing bacteria from a saltmarsh, identifying Novosphingobium sp. MBES04. Experiments showed for the first time that this alphaproteobacterium produces DMSP through the methylation pathway used by plants, not the pathway previously identified in bacteria. Characterization demonstrated DMSP likely serves as an osmoprotectant and is produced more under low nitrogen conditions.
This document outlines a lecture on carbohydrate and nucleic acid chemistry. It begins by introducing carbohydrates as one of the four major classes of biological molecules, along with proteins, lipids, and nucleic acids. Carbohydrates serve important nutritional, structural, informational, and regulatory functions in living systems. They are classified based on their monomer units into monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The lecture further discusses carbohydrate isomers, epimers, enantiomers, cyclization, and polysaccharides such as starch, glycogen, cellulose, and chitin. It then introduces nucleic acids DNA and RNA as polymers of nucleotides, highlighting their monomers, base pairing, and DNA
Metabolism is the sum of all chemical reactions within an organism. Metabolic pathways consist of enzyme-catalyzed reactions organized into linear chains or cyclic pathways. Enzymes lower the activation energy of reactions by binding to substrates and altering their structure. Enzyme inhibitors can competitively or non-competitively bind to enzymes and reduce their activity. Metabolic pathways are regulated by end-product inhibition, which stops pathways once their product reaches a certain concentration. Databases can be used to screen chemicals and identify potential new drugs, like anti-malarial treatments. Rates of enzymatic reactions can be calculated from experimental data and plotted on graphs to determine inhibition type.
1. The study characterized the aggregation of recombinant human Interleukin-1 receptor type II (rhuIL-1RII) using differential scanning calorimetry (DSC) and size exclusion chromatography (SEC).
2. A scan-rate dependence in the DSC experiment and a break from linearity in initial aggregation rates near the melting temperature (Tm) suggested that protein unfolding significantly contributes to the aggregation reaction pathway.
3. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from the irreversibly denatured aggregation process by simulating how unfolding properties could predict aggregation rates at different temperatures above and below the Tm.
This document describes research on using genetically fused gold-binding peptides (GBP1) to direct the self-immobilization of alkaline phosphatase (AP) enzymes on micro-patterned gold substrates. GBP1 was genetically fused to the N-terminus of AP in tandem repeats of 5, 6, 7, and 9 units (nGBP1-AP). The 5GBP1-AP fusion construct displayed the best binding and enzymatic activity. Surface plasmon resonance spectroscopy and atomic force microscopy showed that 5GBP1-AP self-assembled and immobilized on gold surfaces. Immobilized 5GBP1-AP exhibited higher enzymatic activity per area than unmodified AP. This demonstrates the potential
This document describes the total synthesis of bleomycin A2 and related compounds. Two total syntheses of bleomycin A2 are reported that use bleomycin demethyl A2 as a key intermediate. A synthesis of decarbamoyl bleomycin demethyl A2 is also described, which represents the first synthetic access to this bleomycin congener. Improved methods for synthesizing the carbohydrate moiety of bleomycin are explored. Synthetic bleomycin was shown to have the same biological activity and DNA cleavage selectivity as naturally occurring bleomycin.
This document summarizes research on the reduction of manganese porphyrins by flavoenzymes and mitochondrial particles, and how this relates to the catalytic reduction of peroxynitrite. It finds that flavoenzymes like xanthine oxidase and glucose oxidase, as well as mitochondrial Complex I and II, catalyze the reduction of manganese porphyrins using substrates like NADH and succinate. Reduced manganese porphyrins can then rapidly react with peroxynitrite via a two-electron process, producing nitrite and manganese porphyrin radicals. This redox cycle can protect peroxynitrite-sensitive targets in submitochondrial particles.
This document describes a new method for selective N-methylation of peptide backbone amides on resin using the Mitsunobu reaction. The key aspects are:
1) N-trifluoroacetamide (Tfa) was used as the protecting group on resin-bound peptides, which can generate a nucleophilic anion for methylation via Mitsunobu conditions.
2) Tfa-protected peptides on resin underwent efficient (80-99%) and selective N-methylation of the backbone amide using the Mitsunobu reaction with triphenylphosphine, methanol and diisopropyl azodicarboxylate.
3) Unlike other reports, the T
This document discusses enzymes and how they control metabolism in cells. It provides details on how enzymes have an active site that binds to specific substrates, and how enzyme catalysis involves molecular motion and substrate collision with the active site. It describes how temperature, pH, and substrate concentration can affect enzyme activity, and includes graphs to illustrate these relationships. The document discusses enzyme inhibition, denaturation of enzymes, and industrial uses of immobilized enzymes. It also covers the production of lactose-free milk and provides guidance on designing an experiment to test the effects of various factors on enzyme activity.
1) The olsA gene mediates the synthesis of ornithine lipids in P. aeruginosa under phosphate-limiting conditions. Ornithine lipids are phosphate-free and their production allows the bacteria to reduce phosphate utilization in its membranes.
2) Mass spectrometry analysis confirmed the production and structure of ornithine lipids in P. aeruginosa, which contain mainly C16:0 and C18:1 fatty acid chains.
3) While resistance to antimicrobial peptides increases under phosphate limitation, ornithine lipid production was found to not be required for this increased resistance or for virulence in a C. elegans infection model.
1. El documento presenta la agenda del primer día de una asignatura. Incluye la bienvenida a los estudiantes, la presentación del profesor y los objetivos del curso. También cubre las expectativas de los alumnos, el programa, el instructivo académico y una sesión de preguntas y respuestas.
2. La unidad uno trata sobre conceptos básicos de didáctica como aprendizaje, currículo, enseñanza y educación. Explica los tipos de didáctica y los paradigmas educativos.
Este documento presenta el plan de una unidad didáctica para la asignatura Didáctica de la Educación Superior I de una maestría. La unidad uno se enfoca en los fundamentos de la didáctica y analiza conceptos como la problemática didáctica y las soluciones en el proceso educativo superior. Incluye actividades de aprendizaje, evaluación y recursos. La unidad dos examina el proceso didáctico, principios didácticos y características.
Estrategias docentes para un aprendizaje significativo frida barrigaMineducyt El Salvador
Este documento presenta varias estrategias de enseñanza que pueden usarse para promover aprendizajes significativos. Describe estrategias como objetivos, resúmenes, organizadores previos, preguntas intercaladas, ilustraciones y mapas conceptuales. También clasifica las estrategias en preinstruccionales, coinstruccionales y posinstruccionales dependiendo de cuándo se usan, y según los procesos cognitivos que elicitan como activar conocimientos previos o mejorar la comprensión.
La relación entre profesor y alumno debe basarse en el respeto y confianza mutuos. El profesor debe guiar al alumno como sujeto activo en su aprendizaje, creando un ambiente óptimo. Las estrategias motivacionales incluyen conocer los antecedentes de los alumnos, ser claros en las reglas y tareas, y fomentar las relaciones a través de actividades grupales.
El documento describe 10 señales y síntomas comunes de la enfermedad de Alzheimer, como la pérdida de memoria que afecta la vida diaria, la confusión con el tiempo y el lugar, y los problemas con el lenguaje y la toma de decisiones. También detalla conductas relacionadas con la enfermedad, como olvidar citas recientes, repetir preguntas, y tener dificultades para realizar tareas cotidianas. Finalmente, contrasta estas señales con conductas más típicas del envejecimiento normal, como olvidar nombres
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1. Redox-sensitive residue in the actin-binding interface of myosin
Rebecca J. Moen a
, Sinziana Cornea b
, Daniel E. Oseid b
, Benjamin P. Binder b
, Jennifer C. Klein c
,
David D. Thomas b,⇑
a
Department of Chemistry and Geology, Minnesota State University, Mankato, MN 56001, United States
b
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States
c
Department of Biology, University of Wisconsin, Lacrosse, Lacrosse, MN 54601, United States
a r t i c l e i n f o
Article history:
Received 15 September 2014
Available online 26 September 2014
Keywords:
Dictyostelium
Myosin II
Reactive oxygen species (ROS)
Methionine
Methionine sulfoxide reductase (Msr)
Glutathionylation
a b s t r a c t
We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox
regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We
previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394,
near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxi-
dation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr),
restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a
cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model
for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro,
resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methi-
onine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in
both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
To maintain functional homeostasis within cells, molecules
must sensitively detect and respond to the cellular redox state.
Reactive oxygen species (ROS) are a family of oxygen-derived mol-
ecules that cause covalent modification of proteins and DNA. Many
physiological and pathological conditions are associated with the
accumulation of ROS, which are countered by an elaborate system
of antioxidant enzymes and small molecules [1]. An imbalance
between the levels of ROS and antioxidants is generally termed
oxidative stress. The most sensitive and selective targets of ROS
and antioxidants within proteins, with particular relevance to
aging, are the sulfur-containing amino acids, methionine (Met)
and cysteine (Cys) [2].
The rapid rate and reversibility of sulfur oxidation give Met and
Cys important roles as functional redox switches in proteins. For
example, methionine sulfoxide (MetO) can be reduced to Met by
the thioredoxin-dependent enzyme methionine sulfoxide reduc-
tase (Msr). Cys oxidation can result in reversible intra- or intermo-
lecular disulfide formation or other thiol modifications. One such
modification is glutathionylation, which can covalently modify
Cys by attachment of a glutathione (GSH) tripeptide. During epi-
sodes of oxidative stress, when the ratio of oxidized glutathione
(glutathione disulfide, GSSG) to glutathione is high, susceptible
Cys are more likely to be glutathionylated [3–5].
Sulfur-based redox switches are an integral component in
cardiac and skeletal muscle function and regulation [6–10].
ROS-mediated mechanisms fundamental to muscle contractility
include changes in muscle protein gene expression and post-
translational modifications (PTMs) [11,12]. ROS-induced PTMs
can indirectly affect muscle contractility, modifying proteins
involved in calcium handling [13–15], enzymatic signaling
[16,17], or proteolysis [18].
ROS-induced PTMs can also have direct effects on contractility
by modifying sarcomeric proteins [18] such as myosin, the focus
of the present study. Direct site-specific redox modifications to sar-
comeric proteins are emerging as crucial regulators of muscle func-
tion. Met oxidation and glutathionylation in actin results in defects
in polymerization [19] and activation of myosin ATPase activity
[20–23]. Glutathionylation of specific Cys in troponin I and myosin
binding protein C (MyBP-C) alter myofilament sensitivity to
http://dx.doi.org/10.1016/j.bbrc.2014.09.072
0006-291X/Ó 2014 Elsevier Inc. All rights reserved.
Abbreviations: ESI-MS, electrospray ionization mass spectrometry; Dicty, Dicty-
ostelium discoideum; S1dC, Cys-lite catalytic domain of Dicty myosin II; ROS,
reactive oxygen species; Met, methionine; MetO, methionine sulfoxide; Msr,
methionine sulfoxide reductase; Cys, cysteine; GSH, glutathione; GSSG, oxidized
glutathione.
⇑ Corresponding author.
E-mail address: ddt@umn.edu (D.D. Thomas).
Biochemical and Biophysical Research Communications 453 (2014) 345–349
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
2. calcium [24,25]. Site-specific glutathionylation of titin can
modulate muscle elasticity in muscle [26]. Myosin has been
identified as a target of protein oxidation and glutathionylation
[20,22,23,27,28], but little is known about the structural and
functional significance of specific redox sites within myosin.
We have shown previously that site-specific methionine oxida-
tion in the Dicty myosin II catalytic domain causes a functional
decline in actomyosin interaction, correlated with changes in myo-
sin internal dynamics and structure, specifically in the actin-bind-
ing cleft [29]. Oxidation of methionine at position 394 (M394),
directly N-terminal to the cardiomyopathy (CM) loop of myosin,
is responsible for the decline in function [29]. Sequence alignment
of the Dicty myosin II catalytic domain with all human myosin II
isoforms reveals that this residue is a conserved cysteine residue
in all isoforms of human cardiac and skeletal myosin. This con-
served Cys has been identified as a site of glutathionylation in
b-cardiac myosin [27].
In the present study, our goal is to examine directly the redox
sensitivity of this residue. Using site-directed mutagenesis, chem-
ical modification, and redox-specific enzymes, we have produced
and reversed site-specific redox modifications, including Met oxi-
dation and Cys glutathionylation, and determined quantitatively
the associated chemical and functional changes in the myosin cat-
alytic domain. Our results show that myosin is an oxidatively labile
protein whose level of activity can be regulated by the redox status
of selective sulfur-containing functional groups within its catalytic
domain.
2. Methods
2.1. Protein preparations and assays
Site-directed mutagenesis of the Dicty myosin II gene, truncated
at residue 762, containing only a single (non-reactive) cysteine at
position 655 (‘‘Cys-lite’’ S1dC construct [30]), was performed using
the QuikChange II XL kit (Invitrogen). In addition, all constructs
(except for the M394C mutant) contain a T688C mutation (needed
for sufficient protein expression in Dicty) which was spin-labeled
with IASL [4-(2-Iodoacetamido)-2,2,6,6-tetramethyl-1-piperidinyl-
oxy] (Sigma-Aldrich, USA) to protect from oxidative modification.
These proteins were expressed and purified from Dicty orf+ cells
as previously described [31]. F-actin was prepared from rabbit
skeletal muscle as previously described [32,33]. Human methio-
nine sulfoxide reductase A protein was obtained from Abcam Bio-
chemicals (Cambridge, England). Actin-activated ATPase activity
was measured by detection of ADP using an NADH-coupled ATPase
assay [34] using 0.2 lM S1dC, 2 mM ATP, and increasing concen-
trations of phalloidin-stabilized F-actin in 10 mM Tris, 2 mM MgCl2
(pH 7.5). The actin-dependent activity was analyzed to determine
Vmax (specific activity at saturating actin) and KATPase (actin concen-
tration needed for V = 0.5Vmax) [34].
2.2. Reversible oxidation and glutathionylation
Oxidative modification of Met in myosin was accomplished by
treatment with 500 mM hydrogen peroxide (purchased as a
stabilized 30% solution from Sigma–Aldrich) for 30 min on ice, fol-
lowed by dialysis. Reversal of Met oxidation was achieved by
incubating 20 lM myosin for 30 min at 25 °C with 4 lM MsrA
(Abcam) and 1 mM DTT in 10 mM Tris (pH 7.5). Glutathionylation
of the M394C mutant myosin was accomplished by treatment
with varying concentrations of GSH in the presence of 10 mM
diamide for 1 h on ice, followed by dialysis. Glutathionylation
was reversed by addition of 1 mM DTT for 1 h on ice, followed
by dialysis.
2.3. Mass spectrometry
Myosin samples for electrospray ionization mass spectrometry
(ESI-MS) were prepared as previously described [29]. Myosin mass
was determined using a QSTAR quadrupole-TOF mass spectrome-
ter (ABI) with an electrospray ionization source. 20 lM myosin
was introduced into the solvent stream using a 10 lL injection loop
installed in the integrated loop injector with a total of five injec-
tions per sample. ESI spectra were acquired continuously over
the range 500–2000 m/z, and were analyzed with BioAnalyst QS
(ABI) software v 1.1.5.
3. Results
3.1. Site-directed redox modification of myosin residue 394
S1dC, a construct of the myosin II catalytic domain devoid of
reactive cysteines, was used as the model system to examine the
functional consequences of reversible oxidative modifications.
Using Dicty myosin as such a model is justified by its high level
of structural and functional homology with muscle myosin II
[35,36]. S1dC contains nine Met residues, three of which are sus-
ceptible to oxidative modification by H2O2 [29]. Oxidation of Met
394, which is located at the C-terminus of an alpha-helix that tran-
sitions into the cardiomyopathy loop in the actin-binding interface
(Fig. 1A), has been shown to be responsible for the observed func-
tional and structural perturbations. M394 is a cysteine residue in
all isoforms of human cardiac and skeletal myosin (Fig. 1).
Site-directed mutagenesis and site-specific oxidation define the
key redox-sensitive residues that are chemically and functionally
vulnerable to oxidants. Methionine to Leucine (M-to-L) mutations
control susceptibility of Met oxidation, since Leu conservatively
substitutes for Met and is not modified under the conditions used
for in vitro oxidation. Methionine to Glutamine (M-to-Q) mutations
structurally and functionally mimic Met oxidation to Met sulfox-
ide, since the substitution of glutamine for methionine introduces
an oxygen atom at the same position in the side chain as the sulf-
oxide oxygen. This site-directed substitution approach of M-to-L
and M-to-Q has previously been validated for the functional
analysis of site-specific Met oxidation in several protein systems
[37–39]. Dicty mutants M394L and M394Q to were used here to
examine the redox capabilities of this residue. M394 was also
mutated to cysteine residue, as found in various human myosin II
isoforms (Fig. 1B). As all other reactive Cys in S1dC have been
mutated to other residues, this system is ideal for studying site-
specific cysteine modification.
3.2. Reversible oxidation of Met 394
To quantify the use of methionine sulfoxide reductase (Msr) to
repair individual methionine sulfoxides (MetO) in the myosin cat-
alytic domain, we have used in vitro conditions to generate a
homogeneous population of oxidatively modified myosin in which
three of nine Mets are oxidized to their corresponding MetO, as
revealed by mass spectrometry. Prior to oxidative modification,
the Dicty myosin II catalytic domain exhibits one major peak with
a molecular mass of 88,930 Da (Fig. 2A), corresponding to native
S1dC expressed in D. discoideum spin-labeled with IASL. The condi-
tions used for in vitro oxidative modification of myosin result in a
49 Da increase in mass, corresponding to the addition of three oxy-
gen atoms (Fig. 2A) as we have previously shown [29]. Upon treat-
ment of this oxidized myosin with MsrA, the molecular mass of
S1dC decreases by 16 Da, consistent with the reversal of one MetO
back to Met (Fig. 2A). Treatment of native (unoxidized) S1dC with
MsrA shows no shift in molecular mass (Fig. 2A). No additional
346 R.J. Moen et al. / Biochemical and Biophysical Research Communications 453 (2014) 345–349
3. MetO repair is observed following incubation of myosin with MsrA
for longer periods of time.
To determine whether M394 was the MetO reversed to Met by
MsrA, leucine was substituted for methionine at M394 to protect
from oxidation. Oxidation of the M394L mutant with H2O2 caused
a shift of 32 Da, corresponding to the formation of two MetOs
(Fig. 2B), one less than observed in the presence of M394
(Fig. 2A). Treatment of oxidized M394L with MsrA had no effect
on the mass (data not shown), showing that the other oxidized res-
idues cannot be reduced by MsrA. These results, combined with the
results of methionine sulfoxide reversal on globally oxidized Dicty
myosin II, indicate that M394 is the only Met that, after oxidation
to MetO, can be reduced back to Met by MsrA.
Oxidation of myosin with hydrogen peroxide induced a 2-fold
decrease in maximum actin-activated ATPase rate (Vmax) in the
presence of saturating actin, as observed previously [29], and this
inhibition was completely rescued by subsequent treatment with
MsrA (Fig. 2C, M394). Treatment with DTT alone had no effect
(data not shown). Mutation M394Q produced a 2-fold decrease
in actin-activated ATPase activity, matching the effect of oxidation,
and mutation M394L protected myosin against the effects of
oxidation (Fig. 2C).
3.3. Reversible glutathionylation of M394C
ESI-MS was used to quantify the modification of Cys 394 by glu-
tathione (Fig. 3A). Prior to modification, the M394C mutant S1dC
exhibits one major peak with a molecular mass of 88,691
(Fig. 3A). Note the difference from the molecular mass in Fig. 2A
and B due to the absence of spin label. Treatment with glutathione
(GSH) and diamide produced a shift in molecular mass of $305 Da,
corresponding to the addition of 1 glutathione (Fig. 3A, blue to red).
The reversibility of this modification was assessed by addition of
DTT, which caused a shift back to 88,691 (Fig. 3A, red to magenta),
indicating that the modification with glutathione is indeed
reversible.
This glutathionylation treatment produced a substantial
decrease in actin-activated ATPase activity that was reversed by
DTT, with no effect caused by DDT or diamide alone (Fig. 3B and
C). In untreated myosin, Vmax (activity at saturating actin) and
KATPase (actin concentration needed for half-maximal activation)
are 7.0 ± 1.1 sÀ1
and 41.0 ± 11.8 lM; glutathionylation decreased
Vmax by a factor of 5 (to 1.3 ± 0.3 sÀ1
), with no significant effect
on KATPase (52.7 ± 17.5 lM) (Fig. 3D). This result suggests that the
effect of glutathionylation on myosin function is not simply due
to decreased actin affinity, but is due to the reduced activity of
actin-bound myosin.
4. Discussion
We previously identified at least three Dicty myosin II methio-
nines that are susceptible to oxidation by peroxide: M394, M486
and M642, and showed that M394 is responsible for the oxida-
Fig. 1. (A) Structure of the myosin catalytic domain (PDB:1FMV). Light blue: upper
50 kDa domain. Violet: lower 50 kDa domain. Dark blue: N-terminal domain. Teal:
converter domain. Pink: actin-binding loops. Red: nucleotide-binding pocket. Inset:
location of M394 (yellow) N-terminal to the cardiomyopathy (CM) loop (red) in the
myosin actin-binding interface. (B) Sequence alignment of Dicty (Dd) myosin II
M394 residue with Homo sapiens (Hs) skeletal and cardiac muscle myosin II
isoforms (Peri = perinatal, FSkE = fast skeletal embryonic, FSk = fast skeletal, FSk
ex = fast skeletal extraocular, and Ca = cardiac). (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this
article.)
Fig. 2. Chemical and functional reversal of oxidation, using MsrA. (A) ESI-MS of wildtype residue M at position 394. Blue: no treatment. Red: after treatment with H2O2.
Magenta: Red sample after treatment with MsrA. Black: Blue sample after treatment with MsrA. (B) Mutant M394L, same color scheme as A. MsrA treatment had no effect on
the mass of either sample in B (data not shown). (C) Recovery of S1dC actin-activated ATPase activity (Vmax) upon repair of MetO with MsrA. Wildtype S1dC was oxidized with
500 mM H2O2 on ice for 30 min (ox), then treated with 0.5 lM MsrA for 30 min at 25 °C (ox + Msr). The oxido-mimetic mutant M394Q (untreated) is shown for comparison.
Error bars are SEM (n P 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
R.J. Moen et al. / Biochemical and Biophysical Research Communications 453 (2014) 345–349 347
4. tion-induced functional decline in myosin actin-activated ATPase
activity and actomyosin interaction [29]. The present study shows
that the oxidation of M394 is reversible by MsrA and that the
reduction of this MetO completely rescues the decline in ATPase
activity (Fig. 2). Mutation of this Met to Cys, mimicking the residue
present in all human isoforms of skeletal and cardiac myosin,
shows that this Cys can also be oxidatively modified by glutathi-
one, decreasing actomyosin functional interaction (Fig. 3).
Methionine sulfoxide reductase most readily reverses oxidized
methionines that are solvent-exposed and located in protein
regions composed predominantly of random-coil [8,40,41]. It is
therefore not surprising that only oxidation of M394, but not
M486 and M642, is reversible. Based on the crystal structure,
M394 is solvent-exposed when myosin is not bound to actin, and
exists at the extreme end of an alpha-helix, transitioning into a
random coil of the CM-loop (Fig. 1A). Although both M486 and
M642 are also predicted to be solvent-exposed, they exist in highly
structured regions of S1dC, in the force-generating domain. Using
site-directed spin labeling (SDSL) and electron paramagnetic reso-
nance (EPR), we previously showed that oxidation of M394
changes the structural dynamics of the actin-binding cleft, [29].
This change in structural dynamics involves a perturbation of the
actin-binding cleft’s open-to-closed transition, which is necessary
for proper force production and movement [29,42].
Glutathionylation of Cys is a reversible oxidative modification
that plays a key role in redox regulation of proteins and signal
transduction. In -cardiac myosin, three Cys within the myosin
heavy chain are susceptible to glutathionylation [27], which inhib-
its myosin ATPase activity at high levels of GSSG but potentiates
ATPase activity at low levels of GSSG [27]. Glutathionylation of
these three Cys produced changes in the structure of myosin
detected by changes in fluorescence and increased susceptibility
to proteolytic cleavage [27]. Two of these Cys residues, Cys 400
and Cys 695, are located within the myosin catalytic domain.
Cys400 is equivalent to M394 in Dicty myosin, located in the
actin-binding cleft adjacent to the CM-loop, while Cys695 is the
reactive cysteine SH2 in the force-generating domain of myosin.
Our mutagenesis strategy allows for site-specific investigation of
Cys glutathionylation in the myosin catalytic domain. The M394C
S1dC mutant can be modified specifically by glutathione in vitro
(Fig. 3), resulting in a decrease in actin-activated ATPase activity
(Fig. 3). In addition to this direct effect of modifying myosin func-
tion, it is likely that glutathionylation can also play a protective
role [43], protecting myosin against irreversible protein thiol oxi-
dation at Cys residues in crucial subdomains of myosin such as
the actin-binding cleft.
Oxidation of methionine to methionine sulfoxide, as well as glu-
tathionylation of cysteine, has been shown to affect protein sec-
ondary and tertiary structure. The presence of MetO can cause -
helical destabilization [44–48]. Glutathionylation has a similar
effect, having been shown to decrease -helical content of proteins
[49,50]. As both Met oxidation and glutathionylation at residue
394 in Dicty myosin II cause a decrease in actin-activated ATPase
activity of myosin (Figs. 2 and 3), we hypothesize that oxidative
modification at this site, whether Met oxidation or Cys glutath-
ionylation, destabilizes the adjacent helix, changing the structural
dynamics of the entire CM-loop and hence interaction with actin.
This change in structural dynamics at the actin-binding cleft prob-
ably perturbs the weak-to-strong transition of myosin, necessary
for proper force production. This hypothesis will be the subject
of future testing by spectroscopic methods in both muscle and
nonmuscle myosin.
5. Conclusion
Redox regulation has emerged as a crucial modulator of protein
function. The production of ROS in muscle can be due to many
pathophysiological and physiological conditions, including aging,
heart failure, inflammation, and strenuous exercise. ROS-induced
Fig. 3. (A) ESI-MS illustrating glutathionylation and reversal in Dicty myosin II M394C. Blue: untreated. Red: treated with 10 mM GSH plus 10 mM diamide for 1 h on ice.
Magenta: treated with DTT after glutathionylation. (B) ATPase activity of M394C in the presence of 50 lM actin. Untreated (À), after treatment with 10 mM GSH plus 10 mM
diamide, 10 mM GSH plus 10 mM diamide followed by 10 mM DTT, 10 mM DTT alone, or 10 mM diamide alone. Error bars are SEM (n P 3). (C) ATPase activity of M394C
S1dC, in the presence of 50 lM actin, after treatment with increasing concentrations of GSH (with equimolar diamide). (D) Actin-dependence of ATPase activity of M394C,
untreated (closed squares) and treated with 10 mM GSH plus 10 mM diamide (open squares). (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
348 R.J. Moen et al. / Biochemical and Biophysical Research Communications 453 (2014) 345–349
5. oxidative modifications occur frequently in sarcomeric proteins,
including myosin. This work illustrates the potential for myosin
to function as a redox sensor in both nonmuscle and muscle cells,
potentially modulating motility/contractility in response to oxida-
tive stress.
Acknowledgments
We thank Margaret Titus for expert consultation regarding
Dicty myosin. We thank Ivan Rayment for the S1dC plasmid. This
work was supported by NIH grants to DDT (R37 AG026160) and
RJM (F31 AG037303) and from AHA to DEO (12UFEL12040063).
We received excellent assistance with mass spectrometry from
Bruce Witthuhn at the Center for Mass Spectrometry and Proteo-
mics at the University of Minnesota. The Biophysical Spectroscopy
Center and Biomedical Genomics Center at the University of
Minnesota were essential for this work.
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