Site-Specific Binding of Non-Site-Specific Ions.
Ontology highlight
ABSTRACT:
SUBMITTER: Chen SJ
PROVIDER: S-EPMC6588825 | biostudies-literature | 2019 Jun
REPOSITORIES: biostudies-literature
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Project description:The Ca2+-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Ca2+ exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca2+ and several trivalent lanthanide ions) and the carboxylate groups in CaM's EF-hand ion-coordinating sites. Since Tb3+ is commonly used as a luminescent Ca2+ analog in studies of protein-ion binding, it is important to characterize distinctions between the coordination of Ca2+ and the lanthanides in CaM. Although functional assays indicate that Tb3+ fully activates many Ca2+-dependent proteins, our FTIR spectra indicate that Tb3+, La3+, and Lu3+ disrupt the bidentate coordination geometry characteristic of the CaM binding sites' strongly conserved position 12 glutamate residue. The 2D IR spectra indicate that, relative to the Ca2+-bound form, lanthanide-bound CaM exhibits greater conformational flexibility and larger structural fluctuations within its binding sites. Time-dependent 2D IR lineshapes indicate that binding sites in Ca2+-CaM occupy well-defined configurations, whereas binding sites in lanthanide-bound-CaM are more disordered. Overall, the results show that binding to lanthanide ions significantly alters the conformation and dynamics of CaM's binding sites.
| S-EPMC5889669 | biostudies-literature
Project description:Mass spectrometry (MS) can unlock crucial insights into the intricate world of glycosylation analysis. Despite its immense potential, the qualitative and quantitative analysis of isobaric glycopeptide structures remains one of the most daunting hurdles in the field of glycoproteomics. The ability to distinguish between these complex glycan structures poses a significant challenge, hindering our ability to accurately measure and understand the role of glycoproteins in biological systems. A few recent publications described the use of collision energy (CE) modulation to improve structural elucidation, especially for qualitative purposes. Different linkages of glycan units usually demonstrate different stabilities under CID/HCD fragmentation conditions. Fragmentation of the glycan moiety produces low molecular weight ions (oxonium ions) that can serve as a structure-specific signature for specific glycan moieties, however, specificity of these fragments has never been examined closely. Here, we investigated fragmentation specificity using synthetic stable isotope-labelled glycopeptide standards. These standards were isotopically labelled at the reducing terminal GlcNAc, which allowed us to resolve fragments produced by oligomannose core moiety and fragments generated from outer antennary structures. Our research identified the potential for false positive structure assignments due to the occurrence of "Ghost" fragments resulting from single glyco unit rearrangement or mannose core fragmentation within the collision cell. To mitigate this issue, we have established a minimal intensity threshold for these fragments to prevent the misidentification of structure-specific fragments in glycoproteomics analysis. Our findings provide a crucial step forward in the quest for more accurate and reliable glycoproteomics measurements.Graphical abstract
| S-EPMC10245710 | biostudies-literature
Project description:Mass spectrometry (MS) can unlock crucial insights into the intricate world of glycosylation analysis. Despite its immense potential, the qualitative and quantitative analysis of isobaric glycopeptide structures remains one of the most daunting hurdles in the field of glycoproteomics. The ability to distinguish between these complex glycan structures poses a significant challenge, hindering our ability to accurately measure and understand the role of glycoproteins in biological systems. A few recent publications described the use of collision energy (CE) modulation to improve structural elucidation, especially for qualitative purposes. Different linkages of glycan units usually demonstrate different stabilities under CID/HCD fragmentation conditions. Fragmentation of the glycan moiety produces low molecular weight ions (oxonium ions) that can serve as a structure-specific signature for specific glycan moieties; however, the specificity of these fragments has never been examined closely. Here, we particularly focused on N-glycoproteomics analysis and investigated fragmentation specificity using synthetic stable isotope-labeled N-glycopeptide standards. These standards were isotopically labeled at the reducing terminal GlcNAc, which allowed us to resolve fragments produced by the oligomannose core moiety and fragments generated from outer antennary structures. Our research identified the potential for false-positive structure assignments due to the occurrence of "Ghost" fragments resulting from single glyco unit rearrangement or mannose core fragmentation within the collision cell. To mitigate this issue, we have established a minimal intensity threshold for these fragments to prevent misidentification of structure-specific fragments in glycoproteomics analysis. Our findings provide a crucial step forward in the quest for more accurate and reliable glycoproteomics measurements.
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Project description:Site binding of ions and water shapes nucleic acids folding, dynamics, and biological function, complementing the more diffuse, nonspecific "territorial" ion binding. Unlike territorial binding, prediction of site-specific binding to nucleic acids remains an unsolved challenge in computational biophysics. This work presents a new toolset based on the 3D-RISM molecular solvation theory and topological analysis that predicts cation and water site binding to nucleic acids. 3D-RISM is shown to accurately capture alkali cations and water binding to the central channel, transversal loops, and grooves of the Oxytricha nova's telomeres' G-quadruplex ( Oxy-GQ), in agreement with high-resolution crystallographic data. To improve the computed cation occupancy along the Oxy-GQ central channel, it was necessary to refine and validate new cation-oxygen parameters using structural and thermodynamic data available for crown ethers and ion channels. This single set of parameters that describes both localized and delocalized binding to various biological systems is used to gain insight into cation occupancy along the Oxy-GQ channel under various salt conditions. The paper concludes with prospects for extending the method to predict divalent cation binding to nucleic acids. This work advances the forefront of theoretical methods able to provide predictive insight into ion atmosphere effects on nucleic acids function.
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Project description:A new method for identifying residue specific through space contacts as a function of protein secondary and tertiary structure in the gas phase is presented. Photodissociation of a non-native carbon-iodine bond incorporated into Tyr59 of ubiquitin yields a radical site specifically at that residue. The subsequent radical migration is shown to be highly dependent on the structure of the protein. Radical-directed dissociation (RDD) of low charge states, which adopt compact structures, generates backbone fragmentation that is prominently distributed throughout the protein sequence, including residues that are distant in sequence from Tyr59. Higher charge states of ubiquitin, which adopt elongated, unfolded structures, yield RDD that is primarily nearby in sequence to Tyr59. Regardless of which structure is probed, information at the residue-level is obtained by examining specific radical-donor and radical-acceptor pairs. The relative importance of a particular interaction pair for a specific conformation can be revealed by tracking the charge state dependence of the dissociation. Structurally important contact pairs exhibit strong and concerted changes in relative intensities as a function of charge state and can also be used to reveal structural features which persist among different protein structures. Moreover, incorporation of distance constraint information into molecular mechanics conformational searches can be used to drive the search toward relevant conformational space. Implementation of this approach has revealed highly stable, previously undiscovered structures for the +4 and +6 charge states of ubiquitin, which bear little resemblance to the crystal structure.
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Project description:Biological membranes composed of lipids and proteins are in contact with electrolytes like aqueous NaCl solutions. Based on molecular dynamics studies it is widely believed that Na(+) ions specifically bind to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, whereas Cl(-) ions stay in solution. Here, we present a careful comparison of recent data from electrophoresis and isothermal titration calorimetry experiments as well as molecular dynamics simulations suggesting that in fact both ions show very similar affinities. The corresponding binding constants are 0.44(±0.05) M(-1) for Na(+) and 0.40(±0.04) M(-1) for Cl(-) ions. This is highlighted by our observation that a widely used simulation setup showing asymmetric affinities of Na(+) and Cl(-) for POPC bilayers overestimates the effect of NaCl on the electrophoretic mobility of a POPC membrane by an order of magnitude. Implications for previous simulation results on the effect of NaCl on polarization of interfacial water, transmembrane potentials, and mechanisms for ion transport through bilayers are discussed. Our findings suggest that a range of published simulations results on the interaction of NaCl with phosphocholine bilayers have to be reconsidered and revised and that force field refinements are necessary for reliable simulation studies of membranes at physiological conditions on a molecular level.
| S-EPMC3576540 | biostudies-literature
Project description:Programmable nucleases and designer-recombinases are prominent genome editing tools that hold great potential for the treatment of human genetic disorders. However, both of these tools alone are not optimal for clinical applications. We present an approach that combines the ease of targeting of programmable nucleases with editing safety and accuracy of site-specific recombinases. We find that insertional fusions of zinc-finger DNA-binding domains (ZFDs) into the coding sequence of designer-recombinases generate conditional enzymes that are inactive, unless the ZFD binds its target site placed in the vicinity of the recombinase binding site. This induced-fit activity is transferable to a recombinase with relaxed specificity, representing the prototype of a new class of genome editing enzymes that opens exciting perspectives for flexible, seamless, and precise genome surgery.
2023-12-19 | GSE243676 | GEO
Project description:The hepatitis delta virus (HDV) ribozyme uses both metal ion and nucleobase catalysis in its cleavage mechanism. A reverse G·U wobble was observed in a recent crystal structure of the precleaved state. This unusual base pair positions a Mg(2+) ion to participate in catalysis. Herein, we used molecular dynamics (MD) and X-ray crystallography to characterize the conformation and metal binding characteristics of this base pair in product and precleaved forms. Beginning with a crystal structure of the product form, we observed formation of the reverse G·U wobble during MD trajectories. We also demonstrated that this base pair is compatible with the diffraction data for the product-bound state. During MD trajectories of the product form, Na(+) ions interacted with the reverse G·U wobble in the RNA active site, and a Mg(2+) ion, introduced in certain trajectories, remained bound at this site. Beginning with a crystal structure of the precleaved form, the reverse G·U wobble with bound Mg(2+) remained intact during MD simulations. When we removed Mg(2+) from the starting precleaved structure, Na(+) ions interacted with the reverse G·U wobble. In support of the computational results, we observed competition between Na(+) and Mg(2+) in the precleaved ribozyme crystallographically. Nonlinear Poisson-Boltzmann calculations revealed a negatively charged patch near the reverse G·U wobble. This anionic pocket likely serves to bind metal ions and to help shift the pK(a) of the catalytic nucleobase, C75. Thus, the reverse G·U wobble motif serves to organize two catalytic elements, a metal ion and catalytic nucleobase, within the active site of the HDV ribozyme.
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