ABSTRACT
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.
Subject(s)
Rhodopsin , Vision, Ocular , Animals , Binding Sites/radiation effects , Crystallography , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/metabolism , Isomerism , Photons , Protein Binding/radiation effects , Protein Conformation/radiation effects , Retinaldehyde/chemistry , Retinaldehyde/metabolism , Retinaldehyde/radiation effects , Rhodopsin/chemistry , Rhodopsin/metabolism , Rhodopsin/radiation effects , Time Factors , Vision, Ocular/physiology , Vision, Ocular/radiation effectsABSTRACT
Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five 'structural' subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD's DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD's affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62's high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.
Subject(s)
DNA Repair/genetics , RNA-Binding Proteins/ultrastructure , Transcription Factor TFIIH/ultrastructure , Binding Sites/radiation effects , Cryoelectron Microscopy , DNA Damage/radiation effects , DNA Helicases/genetics , DNA Helicases/ultrastructure , DNA, Single-Stranded/genetics , DNA, Single-Stranded/radiation effects , DNA, Single-Stranded/ultrastructure , DNA-Binding Proteins/genetics , DNA-Binding Proteins/ultrastructure , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/ultrastructure , RNA-Binding Proteins/genetics , Single Molecule Imaging , Transcription Factor TFIIH/genetics , Transcription, Genetic/radiation effects , Ultraviolet Rays/adverse effects , Xeroderma Pigmentosum Group D Protein/genetics , Xeroderma Pigmentosum Group D Protein/ultrastructureABSTRACT
DNA-peptide interactions are involved in key life processes, including DNA recognition, replication, transcription, repair, organization, and modification. Development of tools that can influence DNA-peptide binding non-invasively with high spatiotemporal precision could aid in determining its role in cells and tissues. Here, the design, synthesis, and study of photocontrolled tools for sequence-specific small peptide-DNA major and minor groove interactions are reported, shedding light on DNA binding by transcriptionally active peptides. In particular, photoswitchable moieties were implemented in the peptide backbone or turn region. In each case, DNA binding was affected by photochemical isomerization, as determined in fluorescent displacement assays on model DNA strands, which provides promising tools for DNA modulation.
Subject(s)
AT-Hook Motifs , DNA/metabolism , Light , Peptides/metabolism , Zinc Fingers , AT-Hook Motifs/radiation effects , Binding Sites/radiation effects , DNA/chemistry , Isomerism , Models, Molecular , Peptides/chemistry , Photochemical Processes , Zinc Fingers/radiation effectsABSTRACT
The orange carotenoid protein (OCP) plays a key role in cyanobacterial photoprotection. Photoconversion entails structural rearrangements in OCP that are required for its binding to phycobilisome, thereby inducing excitation energy dissipation. Detachment of OCP from phycobilisome requires the fluorescence recovery protein (FRP). It is considered that OCP interacts with FRP only in the photoactivated state; however, the binding site for FRP is currently unknown. As an important stabilizing element in orange OCP, the short αA-helix within the N-terminal extension (NTE) binds to the C-terminal domain (CTD), but unfolds upon photoactivation and interferes with phycobilisome binding. Here, we demonstrate that the NTE shares specific structural and functional similarities with FRP and discover the main site of OCP-FRP interactions in the OCP-CTD.
Subject(s)
Bacterial Proteins/metabolism , Models, Molecular , Phycobilisomes/metabolism , Synechocystis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites/radiation effects , Cross-Linking Reagents/chemistry , Gene Deletion , Hydrogen Bonding , Kinetics , Light , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phycobilisomes/chemistry , Protein Conformation/radiation effects , Protein Conformation, alpha-Helical/radiation effects , Protein Interaction Domains and Motifs , Protein Multimerization/radiation effects , Protein Stability/radiation effects , Protein Unfolding/radiation effects , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Structural Homology, Protein , Synechocystis/enzymologyABSTRACT
Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced.
Subject(s)
1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt/pharmacology , Dermis/drug effects , Fibroblasts/drug effects , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 3/genetics , Transcription Factor AP-1 , Ultraviolet Rays , Antioxidants/pharmacology , Binding Sites/radiation effects , Cells, Cultured , Cytoprotection/drug effects , Cytoprotection/genetics , Cytoprotection/radiation effects , Dermis/cytology , Dermis/metabolism , Dermis/radiation effects , Fibroblasts/metabolism , Fibroblasts/radiation effects , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/radiation effects , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 3/metabolism , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/radiation effects , Signal Transduction/drug effects , Signal Transduction/radiation effects , Skin Aging/drug effects , Skin Aging/radiation effects , Transcription Factor AP-1/metabolism , Transcription Factor AP-1/radiation effects , Transcriptional Activation/drug effects , Transcriptional Activation/radiation effects , Ultraviolet Rays/adverse effectsABSTRACT
The effect of violaxanthin and zeaxanthin, two main carotenoids of the xanthophyll cycle, on molecular organization of LHCII, the principal photosynthetic antenna complex of plants, was studied in a model system based on lipid-protein membranes, by means of analysis of 77 K chlorophyll a fluorescence and "native" electrophoresis. Violaxanthin was found to promote trimeric organization of LHCII, contrary to zeaxanthin which was found to destabilize trimeric structures. Moreover, violaxanthin was found to induce decomposition of oligomeric LHCII structures formed in the lipid phase and characterized by the fluorescence emission band at 715 nm. Both pigments promoted formation of two-component supramolecular structures of LHCII and xanthophylls. The violaxanthin-stabilized structures were composed mostly of LHCII trimers while, the zeaxanthin-stabilized supramolecular structures of LHCII showed more complex organization which depended periodically on the xanthophyll content. The effect of the xanthophyll cycle pigments on molecular organization of LHCII was analyzed based on the results of molecular modeling and discussed in terms of a physiological meaning of this mechanism. Supramolecular structures of LHCII stabilized by violaxanthin, prevent uncontrolled oligomerization of LHCII, potentially leading to excitation quenching, therefore can be considered as structures protecting the photosynthetic apparatus against energy loses at low light intensities.
Subject(s)
Light-Harvesting Protein Complexes/chemistry , Molecular Docking Simulation , Photosystem II Protein Complex/chemistry , Zeaxanthins/chemistry , Binding Sites/radiation effects , Light , Light-Harvesting Protein Complexes/radiation effects , Photosystem II Protein Complex/radiation effects , Protein Binding/radiation effects , Protein Conformation/radiation effects , Radiation Dosage , Xanthophylls/chemistry , Xanthophylls/radiation effects , Zeaxanthins/radiation effectsABSTRACT
Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecule, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G ⢠U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G ⢠U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.
Subject(s)
DNA/chemistry , Photosensitizing Agents/chemistry , RNA/chemistry , Riboflavin/chemistry , Base Sequence/radiation effects , Binding Sites/radiation effects , DNA/radiation effects , Light , Molecular Sequence Data , Photosensitizing Agents/radiation effects , RNA/radiation effects , Riboflavin/radiation effects , Structure-Activity RelationshipABSTRACT
In this work, the behavior of protein molecules adsorbed on TiO2 nanodots films are modulated through the light responses of the nanodots. TiO2 nanodots films are first prepared through phase separation induced self assembly. Then, bovine serum albumin (BSA) is adsorbed on TiO2 nanodots films and exposed to ultraviolet (365 nm) illumination. It is found the conformation of surface-bound BSA molecules changes with ultraviolet illumination. Moreover, the BSA molecules conjugate to the surface-bound molecules, which are in the overlayer, are released. The reason is ascribed to that TiO2 nanodots absorb ultraviolet and result in the increase of surface hydroxyl groups on nanodots. Such increase further leads to intensified attraction of -NH3 groups in the surface-bound BSA molecules. That not only changes the conformation of the surface-bound BSA molecules, but also weaken the conjugation between surface-bound molecules and other BSA molecules in the overlayer. Eventually, the overlayer of BSA molecules is released. It is believed that such protein conformation variation and release behavior induced through light responses of TiO2 nanodots are crucial in understanding the biomedical performance of TiO2 nanostructures. Also, it could be widely utilized in tailoring of the materials-protein interactions.
Subject(s)
Membranes, Artificial , Metal Nanoparticles/chemistry , Metal Nanoparticles/radiation effects , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/radiation effects , Titanium/chemistry , Adsorption/radiation effects , Binding Sites/radiation effects , Light , Materials Testing , Particle Size , Protein Binding/radiation effects , Protein Conformation/radiation effects , Radiation Dosage , Surface Properties , Titanium/radiation effects , Ultraviolet RaysABSTRACT
The xeroderma pigmentosum group G (XPG) gene, encoding an essential element in nucleotide excision repair (NER), has a proliferating cell nuclear antigen-binding domain (PCNA-BD) at its C-terminal region. However, the role of this domain is controversial because its presence does not affect NER. Using yeast RAD2, a homolog of human XPG, we show that Rad2p interacts with PCNA through its PCNA-BD and the PCNA-BD of Rad2p plays a role in UV-induced mutagenesis. While a mutation of Rad2p endonuclease activity alone causes dramatically increased mutation rates and UV sensitivity, as well as growth retardation after UV irradiation, a mutation of the Rad2p PCNA-BD in the same mutant causes dramatically decreased mutation rates, reduced UV sensitivity and increased growth rate after UV irradiation. After UV irradiation, large-budded cells of Rad2p endonuclease defective mutants wane due to a mutation of the Rad2p PCNA-BD. Besides, the Rad2p PCNA-BD mutant protein exhibits alleviated PCNA-binding efficiency. These results show a hitherto unsuspected role of the Rad2p PCNA-BD that controls mutagenesis via cell cycle modulation together with PCNA. Furthermore, the high mutation rate of cells with other NER gene mutations was also decreased by the mutation of the Rad2p PCNA-BD, which indicates that the Rad2p-PCNA interaction might be responsible for mutagenesis control in the general NER pathway. Our results suggest that the drastically increased incidence of skin cancer in xeroderma pigmentosum patients could arise from the synergistic effects between cell cycle arrest due to the XPG-PCNA interaction and the accumulation of damaged DNA via defects in DNA damage repair.
Subject(s)
Cell Cycle , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/radiation effects , Binding Sites/radiation effects , Cell Cycle/radiation effects , DNA Damage/radiation effects , DNA Repair/radiation effects , Endodeoxyribonucleases/chemistry , Gene Expression Regulation, Fungal/radiation effects , Mutagenesis/radiation effects , Mutation Rate , Proliferating Cell Nuclear Antigen/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymologyABSTRACT
It has been 50 years since F. H. Westheimer and colleagues reported the first use of a photoactivatable cross-linking reagent to study the active site of chymotrypsin. In studies of seven transmembrane helical receptors, also known as G protein-coupled receptors (GPCRs), recent simultaneous advances in structural biology, molecular dynamics simulations, and amber codon suppression methods have allowed the development of a targeted photo-cross-linking strategy to probe receptor-ligand interactions in cell membranes. We review here recent advances in targeted photo-cross-linking of GPCR-ligand complexes in the context of extensive earlier work that primarily relied upon the use of ligand analogues with photoactivatable constituents.
Subject(s)
Cross-Linking Reagents/chemistry , Photoaffinity Labels/chemistry , Protein Interaction Mapping/methods , Receptors, G-Protein-Coupled/chemistry , Binding Sites/drug effects , Binding Sites/radiation effects , Humans , Ligands , Photochemical Processes , Protein Binding/drug effects , Protein Binding/radiation effects , Structure-Activity RelationshipABSTRACT
A genetically encoded optogenetic system was constructed that activates mRNA translation in mammalian cells in response to light. Blue light induces the reconstitution of an RNA binding domain and a translation initiation domain, thereby activating target mRNA translation downstream of the binding sites.
Subject(s)
Protein Biosynthesis/radiation effects , RNA, Messenger/metabolism , Binding Sites/radiation effects , HEK293 Cells , Humans , LightABSTRACT
In order to investigate the binding ability of Lactobacillus strains to Benzo(a)pyrene (BaP), 15 strains were analysed. L. plantarum CICC 22135 and L. pentosus CICC 23163 exhibited high efficiency in removing BaP from aqueous medium; the binding rates were 66.76% and 64.31%, respectively. This process was affected by temperature, incubation time and pH, and cell viability was not necessary for the binding ability. Additionally, both strains, especially strain CICC 23163 showed high specificity in binding BaP. The cell-BaP complexes were stable in aqueous medium. The mechanism of binding was investigated by examining the binding ability of different components of the microorganism cells. The results revealed that peptidoglycans played an important role in binding BaP and its structural integrity was required. Consequently, we proposed that the mechanism of this process was a physisorption and peptidoglycan was the main binding site. These two strains may be used for dietary detoxification in human diet and animal feed.
Subject(s)
Bacterial Proteins/metabolism , Benzo(a)pyrene/metabolism , Carcinogens/metabolism , Food Contamination/prevention & control , Lactobacillus/metabolism , Peptidoglycan/metabolism , Adsorption/radiation effects , Bacterial Proteins/analysis , Bacterial Proteins/chemistry , Bacterial Proteins/radiation effects , Benzo(a)pyrene/toxicity , Binding Sites/radiation effects , Carcinogens/toxicity , Cell Wall/chemistry , Cell Wall/drug effects , Cell Wall/metabolism , Hydrogen-Ion Concentration , Kinetics , Lactobacillus/drug effects , Lactobacillus/growth & development , Lactobacillus/radiation effects , Lactobacillus plantarum/drug effects , Lactobacillus plantarum/growth & development , Lactobacillus plantarum/metabolism , Lactobacillus plantarum/radiation effects , Membrane Glycoproteins/analysis , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Microbial Viability , Peptidoglycan/analysis , Peptidoglycan/chemistry , Peptidoglycan/radiation effects , Protein Stability/radiation effects , Sonication , Spheroplasts/chemistry , Spheroplasts/drug effects , Spheroplasts/metabolism , Surface Properties/radiation effects , Teichoic Acids/analysis , Teichoic Acids/metabolism , TemperatureABSTRACT
The use of density functional theory (DFT) and time-dependent DFT (TD-DFT) to study the photochemistry of metal complexes is becoming increasingly important among chemists. Computational methods provide unique information on the electronic nature of excited states and their atomic structure, integrating spectroscopy observations on transient species and excited-state dynamics. In this contribution, we present an overview on photochemically active transition metal complexes investigated by DFT. In particular, we discuss a representative range of systems studied up to now, which include CO- and NO-releasing inorganic and organometallic complexes, haem and haem-like complexes dissociating small diatomic molecules, photoactive anti-cancer Pt and Ru complexes, Ru polypyridyls and diphosphino Pt derivatives.
Subject(s)
Models, Chemical , Models, Molecular , Photochemistry/methods , Transition Elements/chemistry , Transition Elements/radiation effects , Binding Sites/radiation effects , Computer Simulation , Light , Molecular Conformation/radiation effectsABSTRACT
A peptide containing a single disulfide bond was sequenced using high-energy collision-induced dissociation (HE-CID) in conjunction with a high mass resolution time-of-flight tandem mass spectrometer equipped with a matrix-assisted laser desorption/ionization source. This mass spectrometer, which has spiral ion trajectory, allowed both high mass resolution and high precursor ion selectivity. It is difficult to obtain sufficient product ions from peptides containing disulfide bonds using HE-CID due to the single collision in the gas phase. To compensate for insufficient dissociation, the disulfide bond was cleaved via an in-source reduction process using 1,5-diaminonaphthalene, a reducing matrix. After applying the reduction in the ionization, subsequent sequencing using HE-CID provided the detailed structural information of the peptide containing the single disulfide bond.
Subject(s)
Disulfides/chemistry , Mass Spectrometry/methods , Peptide Mapping/methods , Peptides/chemistry , Peptides/radiation effects , Sequence Analysis, Protein/methods , Amino Acid Sequence , Binding Sites/radiation effects , Disulfides/radiation effects , Heavy Ions , Molecular Sequence Data , Protein Binding/radiation effectsABSTRACT
This work describes an in vivo study analyzing the effect of acoustic radiation force (ARF) on the binding of BR55 VEGFR2-specific contrast-agent microbubbles in a model of prostatic adenocarcinoma in rat. A commercial ultrasound system was modified by implementing high duty-cycle 3.5-MHz center frequency ARF bursts in a scanning configuration. This enabled comparing the effects of ARF on binding in tumor and healthy tissue effectively in the same field of view. Bubble binding was established by measuring late-phase enhancement in amplitude modulation (AM) contrast-specific imaging mode (4 MHz, 150 kPa) 10 min after agent injection when the unbound bubbles were cleared from the circulation. Optimal experimental conditions, such as agent concentration (0.4 × 10(8)-1.6 × 10(8) bubbles/kg), acoustic pressure amplitude (26-51 kPa) and duty-cycle (20%-95%) of the ARF bursts, were evaluated in their ability to enhance binding in tumor without significantly increasing binding in healthy tissue. Using the optimal conditions (38 kPa peak-negative pressure, 95% duty cycle), ARF-assisted binding of BR55 improved significantly in tumor (by a factor of 7) at a lower agent dose compared with binding without ARF, and it had an insignificant effect on binding in healthy tissue. Thus, the high binding specificity of BR55 microbubbles for targeting VEGFR2 present at sites of active angiogenesis was confirmed by this study. Therefore, it is believed that based on the results obtained in this work, ultrasound molecular imaging using target-specific contrast-agent microbubbles should preferably be performed in combination with ARF.
Subject(s)
Adenocarcinoma/diagnostic imaging , Adenocarcinoma/metabolism , Contrast Media/pharmacokinetics , Elasticity Imaging Techniques/methods , Prostatic Neoplasms/diagnostic imaging , Prostatic Neoplasms/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Binding Sites/radiation effects , Cell Line, Tumor , Drug Delivery Systems/methods , High-Energy Shock Waves , Male , Microbubbles , Protein Binding/radiation effects , Rats , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
In this study, a new small-molecule-based reagent was designed to recognize and bind to specific site in protein. A new pyrenyl probe, d-biotinyl-1(1-pyrene)methylamide (Py-biotin) was designed and synthesized by coupling of d-biotin to 1(1-pyrene)methylamine hydrochloride. Binding studies and site-specific photocleavage of avidin by Py-biotin were demonstrated. Binding of Py-biotin to avidin was studied using absorbance and fluorescence spectroscopic techniques. Red shifts of the absorption peak positions of the pyrenyl chromophore followed by hyperchromism were observed upon binding to avidin. The photocleavage of avidin was achieved when a mixture of the protein, Py-biotin, and an electron acceptor, cobalt(III) hexammine trichloride (CoHA), was irradiated at 342nm. No reaction occurred in the absence of the probe, CoHA, or light. N-terminal sequencing of the peptide fragments indicated a cleavage site of avidin between Thr 77 and Val 78. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.
Subject(s)
Avidin/radiation effects , Biotin/analogs & derivatives , Biotin/chemical synthesis , Fluorescent Dyes/chemical synthesis , Light , Photolysis , Pyrenes/chemical synthesis , Absorption , Avidin/metabolism , Binding Sites/radiation effects , Biotin/chemistry , Chlorides/metabolism , Cobalt/metabolism , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes/chemistry , Peptide Fragments/metabolism , Peptide Fragments/radiation effects , Pyrenes/chemistry , Spectrometry, Fluorescence , Threonine/metabolism , Valine/metabolismABSTRACT
A metal ion (Cu(2+)) and a complex copper species, copper (II) bis-bipyridine, were used as alternate counterions in an aqueous surfactant solution of sodium dodecylbenzenesulfonate (SDBS) to investigate the dynamics of counterion interactions in an acoustic field. Sonoluminescence spectral studies showed that such counterions were able to replace sodium ions at the interface, even when the interface was rapidly oscillating under the acoustic field. Ultrasound induced nebulisation was then used to probe the interfacial profile of surfactant and bound counterions in a dynamic environment. At low bulk concentrations, the copper (II) bis-bipyridine cation was more effective at enhancing the loading of the dodecylbenzenesulfonate anion on the interface, due to its documented greater binding ability. However, at higher bulk concentrations, the movement of this cation is limited by its larger size and the smaller Cu(2+) cation is more effective in enhancing the loading of the dodecylbenzenesulfonate anion. The results show that under dynamic conditions, the surface concentrations are governed by mass transfer kinetics rather than equilibrium thermodynamics.
Subject(s)
Copper/chemistry , Copper/radiation effects , Gases/chemical synthesis , Gases/radiation effects , Sonication/methods , Surface-Active Agents/chemistry , Surface-Active Agents/radiation effects , Binding Sites/radiation effects , Ions , Radiation DosageABSTRACT
Blue-light sensitive photoreceptory BLUF domains are flavoproteins, which regulate various, mostly stress-related processes in bacteria and eukaryotes. The photoreactivity of the flavin adenine dinucleotide (FAD) cofactor in three BLUF domains from Rhodobacter sphaeroides, Synechocystis sp. PCC 6803 and Escherichia coli have been studied at low temperature using time-resolved electron paramagnetic resonance. Photoinduced flavin triplet states and radical-pair species have been detected on a microsecond time scale. Differences in the electronic structures of the FAD cofactors as reflected by altered zero-field splitting parameters of the triplet states could be correlated with changes in the amino-acid composition of the various BLUF domains' cofactor binding pockets. For the generation of the light-induced, spin-correlated radical-pair species in the BLUF domain from Synechocystis sp. PCC 6803, a tyrosine residue near the flavin's isoalloxazine moiety plays a critical role.
Subject(s)
Bacterial Proteins/metabolism , Flavin-Adenine Dinucleotide/metabolism , Flavins/metabolism , Flavoproteins/metabolism , Photoreceptors, Microbial/metabolism , Tyrosine/metabolism , Bacterial Proteins/chemistry , Binding Sites/radiation effects , Electron Spin Resonance Spectroscopy , Electrons , Escherichia coli/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavoproteins/chemistry , Free Radicals , Light , Photochemical Processes/radiation effects , Photoreceptors, Microbial/chemistry , Protein Binding/radiation effects , Protein Structure, Tertiary/radiation effects , Rhodobacter sphaeroides/metabolism , Synechocystis/metabolismABSTRACT
The cohesin complex plays a central role in genome maintenance by regulation of chromosome segregation in mitosis and DNA damage response (DDR) in other phases of the cell cycle. The ATM/ATR phosphorylates SMC1 and SMC3, two core components of the cohesin complex to regulate checkpoint signaling and DNA repair. In this report, we show that the genome-wide binding of SMC1 and SMC3 after ionizing radiation (IR) is enhanced by reinforcing pre-existing cohesin binding sites in human cancer cells. We demonstrate that ATM and SMC3 phosphorylation at Ser(1083) regulate this process. We also demonstrate that acetylation of SMC3 at Lys(105) and Lys(106) is induced by IR and this induction depends on the acetyltransferase ESCO1 as well as the ATM/ATR kinases. Consistently, both ESCO1 and SMC3 acetylation are required for intra-S phase checkpoint and cellular survival after IR. Although both IR-induced acetylation and phosphorylation of SMC3 are under the control of ATM/ATR, the two forms of modification are independent of each other and both are required to promote reinforcement of SMC3 binding to cohesin sites. Thus, SMC3 modifications is a mechanism for genome-wide reinforcement of cohesin binding in response to DNA damage response in human cells and enhanced cohesion is a downstream event of DDR.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Genome, Human/radiation effects , Acetylation/radiation effects , Animals , Ataxia Telangiectasia Mutated Proteins , Binding Sites/radiation effects , Chondroitin Sulfate Proteoglycans/metabolism , Chromatin/metabolism , Chromatin/radiation effects , DNA Breaks, Double-Stranded/radiation effects , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Phosphorylation/radiation effects , Protein Binding/radiation effects , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , CohesinsABSTRACT
The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that gamma-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604-612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions.