Mechanism Of Ion Binding Research Articles

NMR investigations on Cl− and Na+ ion binding during the early hydration process of C3S, C3A and cement paste: A combined modelling and experimental study

Using materials containing sodium chloride(e.g., seawater) to make concrete is sometimes unavoidable. However, the associated ion binding mechanisms and structural evolution during the early hydration process are not fully understood. This study investigated Cl− and Na + binding behaviour and microstructure of C3S, C3A and cement paste within the first 48h using a high-field NMR setup. Results show that during hydration, KOH can reduce Cl− binding but enhance Na + ion binding in C3S paste, while increasing gypsum dosage in C3A paste retards both Cl− and Na+ binding. Retarders reduce Cl− and Na+ binding, while accelerators slightly promote Cl− binding but reduce Na+ binding in cement paste. It is also found that boundary nucleation and growth(BNG) modelling is the most suitable for fitting the ion binding. Finally, the microstructure assessed by the T1 relaxation rate is related to ion binding in various ranges, indicating that hydrates during hydration have various binding efficiencies.

Molecular Simulation of Chloride Ion Binding Mechanisms to Na-Doped Tobermorite 14 Å as a Model System for Sodium-Containing Cements

Molecular Simulation of Chloride Ion Binding Mechanisms to Na-Doped Tobermorite 14 Å as a Model System for Sodium-Containing Cements

Molecular Insights into the Calcium Binding in Troponin C through a Molecular Dynamics Study

Calcium-binding proteins play critical roles in various biological processes such as signal transduction, cell growth, and transcription factor regulation. Ion binding and target binding of Ca2+-binding proteins are highly related. Therefore, understanding the ion binding mechanism will benefit the relevant inhibitor design toward the Ca2+-binding proteins. The EF-hand is the typical ion binding motif in Ca2+-binding proteins. Previous studies indicate that the ion binding affinity of the EF-hand increases with the peptide length, but this mechanism has not been fully understood. Herein, using molecular dynamics simulations, thermodynamic integration calculations, and molecular mechanics Poisson-Boltzmann surface area analysis, we systematically investigated four Ca2+-binding peptides containing the EF-hand loop in site III of rabbit skeletal troponin C. These four peptides have 13, 21, 26, and 34 residues. Our simulations reproduced the observed trend that the ion binding affinity increases with the peptide length. Our results implied that the E-helix motif preceding the EF-hand loop, likely the Phe99 residue in particular, plays a significant role in this regulation. The E-helix has a significant impact on the backbone and side-chain conformations of the Asp103 residue, rigidifying important hydrogen bonds in the EF-hand and decreasing the solvent exposure of the Ca2+ ion, hence leading to more favorable Ca2+ binding in longer peptides. The present study provides molecular insights into the ion binding in the EF-hand and establishes an important step toward elucidating the responses of Ca2+-binding proteins toward the ion and target availability.

Thiacalixarenes with Sulfur Functionalities at Lower Rim: Heavy Metal Ion Binding in Solution and 2D-Confined Space.

Sulfur-containing groups preorganized on macrocyclic scaffolds are well suited for liquid-phase complexation of soft metal ions; however, their binding potential was not extensively studied at the air–water interface, and the effect of thioether topology on metal ion binding mechanisms under various conditions was not considered. Herein, we report the interface receptor characteristics of topologically varied thiacalixarene thioethers (linear bis-(methylthio)ethoxy derivative L2, O2S2-thiacrown-ether L3, and O2S2-bridged thiacalixtube L4). The study was conducted in bulk liquid phase and Langmuir monolayers. For all compounds, the highest liquid-phase extraction selectivity was revealed for Ag+ and Hg2+ ions vs. other soft metal ions. In thioether L2 and thiacalixtube L4, metal ion binding was evidenced by a blue shift of the band at 303 nm (for Ag+ species) and the appearance of ligand-to-metal charge transfer bands at 330–340 nm (for Hg2+ species). Theoretical calculations for thioether L2 and its Ag and Hg complexes are consistent with experimental data of UV/Vis, nuclear magnetic resonance (NMR) spectroscopy, and single-crystal X-ray diffractometry of Ag–thioether L2 complexes and Hg–thiacalixtube L4 complex for the case of coordination around the metal center involving two alkyl sulfide groups (Hg2+) or sulfur atoms on the lower rim and bridging unit (Ag+). In thiacrown L3, Ag and Hg binding by alkyl sulfide groups was suggested from changes in NMR spectra upon the addition of corresponding salts. In spite of the low ability of the thioethers to form stable Langmuir monolayers on deionized water, one might argue that the monolayers significantly expand in the presence of Hg salts in the water subphase. Hg2+ ion uptake by the Langmuir–Blodgett (LB) films of ligand L3 was proved by X-ray photoelectron spectroscopy (XPS). Together, these results demonstrate the potential of sulfide groups on the calixarene platform as receptor unit towards Hg2+ ions, which could be useful in the development of Hg2+-selective water purification systems or thin-film sensor devices.

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

Electron paramagnetic resonance (EPR) spectroscopy has emerged as an ideal biophysical tool to study complex biological processes. EPR spectroscopy can follow minor conformational changes in various proteins as a function of ligand or protein binding or interactions with high resolution and sensitivity. Resolving cellular mechanisms, involving small ligand binding or metal ion transfer, is not trivial and cannot be studied using conventional biophysical tools. In recent years, our group has been using EPR spectroscopy to study the mechanism underlying copper ion transfer in eukaryotic and prokaryotic systems. This mini-review focuses on our achievements following copper metal coordination in the diamagnetic oxidation state, Cu(I), between biomolecules. We discuss the conformational changes induced in proteins upon Cu(I) binding, as well as the conformational changes induced in two proteins involved in Cu(I) transfer. We also consider how EPR spectroscopy, together with other biophysical and computational tools, can identify the Cu(I)-binding sites. This work describes the advantages of EPR spectroscopy for studying biological processes that involve small ligand binding and transfer between intracellular proteins.

Fast and efficient cadmium biosorption by Chlorella vulgaris K-01 strain: The role of cell walls in metal sequestration

In this study a newly identified algal strain, isolated from a saline synanthropic environment and identified as Chlorella vulgaris has been investigated in terms of its physiological responses to elevated cadmium concentrations. Furthermore, cadmium binding capacity and mechanisms that underlie of the cadmium biosorption by these Chlorella vulgaris K-01 cells are described. The mechanism of ion binding is based on chemisorption with a high initial sorption rate. The capacity of Cd2+ removal by living biomass of Chlorella vulgaris K-01 was found to be independent of initial pH over a broad pH range (6.0–9.0). The subsequent FTIR and XPS analyses demonstrated a crucial role of cell wall components containing amino and carboxyl residues in the sorption of cadmium ions. Chlorella vulgaris K-01 can withstand Cd2+ concentrations up to 44.5 μM without significant oxidative stress symptoms and without photosynthesis inhibition impairing its proliferative activity. Chlorella vulgaris K-01 also tolerated an elevated salinity up to 4% NaCl. We conclude that Chlorella vulgaris K-01 possesses substantial potential in cadmium adsorption, suitable for industrial scale applications.

Frontispiz: Universality of the Sodium Ion Binding Mechanism in Class A G‐Protein‐Coupled Receptors

Frontispiz: Universality of the Sodium Ion Binding Mechanism in Class A G‐Protein‐Coupled Receptors

Frontispiece: Universality of the Sodium Ion Binding Mechanism in Class A G-Protein-Coupled Receptors

Frontispiece: Universality of the Sodium Ion Binding Mechanism in Class A G-Protein-Coupled Receptors

Universality of the Sodium Ion Binding Mechanism in Class A G‐Protein‐Coupled Receptors

AbstractThe allosteric modulation of G‐protein‐coupled receptors (GPCRs) by sodium ions has received significant attention as crystal structures of several receptors show Na+ ions bound to the inactive conformations at the conserved Asp2.50. To date, structures from 24 families of GPCRs have been determined, though mechanistic insights into Na+ binding to the allosteric site are limited. We performed hundreds‐of‐microsecond long simulations of 18 GPCRs and elucidated their Na+ binding mechanism. In class A GPCRs, the Na+ ion binds to the conserved residue 2.50 whereas in class B receptors, it binds at 3.43b, 6.53b, and 7.49b. Using Markov state models, we obtained the free energy profiles and kinetics of Na+ binding to the allosteric site, which reveal a conserved mechanism of Na+ binding for GPCRs and show the residues that act as major barriers for ion diffusion. Furthermore, we also show that the Na+ ion can bind to GPCRs from the intracellular side when the allosteric site is inaccessible from the extracellular side.

Universality of the Sodium Ion Binding Mechanism in Class A G-Protein-Coupled Receptors.

The allosteric modulation of G-protein-coupled receptors (GPCRs) by sodium ions has received significant attention as crystal structures of several receptors show Na+ ions bound to the inactive conformations at the conserved Asp2.50 . To date, structures from 24 families of GPCRs have been determined, though mechanistic insights into Na+ binding to the allosteric site are limited. We performed hundreds-of-microsecond long simulations of 18 GPCRs and elucidated their Na+ binding mechanism. In class A GPCRs, the Na+ ion binds to the conserved residue 2.50 whereas in class B receptors, it binds at 3.43b, 6.53b, and 7.49b. Using Markov state models, we obtained the free energy profiles and kinetics of Na+ binding to the allosteric site, which reveal a conserved mechanism of Na+ binding for GPCRs and show the residues that act as major barriers for ion diffusion. Furthermore, we also show that the Na+ ion can bind to GPCRs from the intracellular side when the allosteric site is inaccessible from the extracellular side.

Salt-bridge modulates differential calcium-mediated ligand binding to integrin \u03b11- and \u03b12-I domains

Integrins are transmembrane cell-extracellular matrix adhesion receptors that impact many cellular functions. A subgroup of integrins contain an inserted (I) domain within the α–subunits (αI) that mediate ligand recognition where function is contingent on binding a divalent cation at the metal ion dependent adhesion site (MIDAS). Ca2+ is reported to promote α1I but inhibit α2I ligand binding. We co-crystallized individual I-domains with MIDAS-bound Ca2+ and report structures at 1.4 and 2.15 Å resolution, respectively. Both structures are in the “closed” ligand binding conformation where Ca2+ induces minimal global structural changes. Comparisons with Mg2+-bound structures reveal Mg2+ and Ca2+ bind α1I in a manner sufficient to promote ligand binding. In contrast, Ca2+ is displaced in the α2I domain MIDAS by 1.4 Å relative to Mg2+ and unable to directly coordinate all MIDAS residues. We identified an E152-R192 salt bridge hypothesized to limit the flexibility of the α2I MIDAS, thus, reducing Ca2+ binding. A α2I E152A construct resulted in a 10,000-fold increase in Mg2+ and Ca2+ binding affinity while increasing binding to collagen ligands 20%. These data indicate the E152-R192 salt bridge is a key distinction in the molecular mechanism of differential ion binding of these two I domains.

Mechanism and Energetics of Ion and Tetrodotoxin Binding to NavMs Channel

Voltage-gated sodium channels (Nav) are important targets for treating various diseases. Crystal structure of the bacterial voltage-gated sodium channel NavMs in the open conformation has been obtained by Ulmschneider et al recently. We used this structure in our simulation work in order to study channel stability, sodium ion coordination as well as the ion permeation mechanism. We have employed free energy techniques to calculate the potential of mean force (PMF) for ion movement through the NavMs channel.

NMR Structures and Dynamics in a Prohead RNA Loop that Binds Metal Ions

Metal ions are critical for RNA structure and enzymatic activity. We present the structure of an asymmetric RNA loop that binds metal ions and has an essential function in a bacteriophage packaging motor. Prohead RNA is a noncoding RNA that is required for genome packaging activity in phi29-like bacteriophage. The loops in GA1 and phi29 bacteriophage share a conserved adenine that forms a base triple, although the structural context for the base triple differs. NMR relaxation studies and femtosecond time-resolved fluorescence spectroscopy reveal the dynamic behavior of the loop in the metal ion bound and unbound forms. The mechanism of metal ion binding appears to be an induced conformational change between two dynamic ensembles rather than a conformational capture mechanism. These results provide experimental benchmarks for computational models of RNA-metal ion interactions.

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca2+ binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca2+ is essential for stability as well as correct folding of the whole protein. In the absence of Ca2+, pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca2+ addition, indicating that these are not dead-end species and that Ca2+ drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6–7M urea, relates to Ca2+ dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca2+ induced folding is not restricted to the RTX domain. Taken together, Ca2+ binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation.

Cation Bridging Studied by Specular Neutron Reflection

The binding of an anionic surfactant onto an anionic surface by addition of divalent ions is reported based on experimental data from specular neutron reflection (NR) and attenuated total internal reflection IR spectroscopy (ATR-IR). Similar measurements using monovalent ions (sodium) do not show any evidence of such adsorption, even though the amount of surfactant can be much higher. This data is interpreted in terms of the so-called bridging mechanism of ion binding.

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ~1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.

Insights into the Mechanism of Metal Ion Binding to an EF-Hand from the X-ray Structure of Mg(2+)-Bound N-Domain of Calmodulin

The intracellular concentration of Mg2+ (∼1.0 mM) is sufficiently high to occupy the Ca2+-binding sites in the N-domain of calmodulin (N-CaM) at the resting Ca2+ levels. Thus, Mg2+ is expected to modulate the intracellular function of CaM. Here we report the 2.1 A resolution X-ray structure of the Mg2+-bound N-CaM. The complex was crystallized in P1 space group with two molecules in the asymmetric unit arranged into a back-to-back dimer. Both N-CaM monomers adopt the closed-domain (off-state) conformation. The structure reveals two modes of metal binding to an EF-hand. In site I Mg2+ occupies the usual metal binding position engaging directly the ligands typically involved in Ca2+ coordination, with the exception of the bidentate coordination from the glutamate side chain in the 12th position. The absence of that Ca2+-specific interaction explains the closed domain conformation. Unexpectedly, in site II Mg2+ contacts the carbonyl oxygen of the last residue in the entering helix (X-1 position) and the central carbonyl oxygen of the loop (-Y position), which is a part of the EF-hand-β-scaffold. The remaining four coordinating positions are occupied by water, which, in turn, make strong two-pronged hydrogen bonds to sidechain carboxyls of Asp and Glu in Y and -Z positions of the loop, respectively. This unusual binding mode is apparently facilitated by a shift of the entering helix stabilized by the protein interface in the crystal lattice. The structure offers a view to the mechanism of an initial engagement of a hydrated metal ion with an EF-hand and a plausible route for its dehydration and transfer to the final binding position. These observations support and extend our previously proposed two-step EFBS mechanism (Grabarek, Z., J. Mol. Biol. 346,1351,2005).

Molecular Mechanisms of Ion Selectivity in Membrane Proteins: Ion Channels and Transporters

The mechanism of ion binding to membrane proteins and subsequent ion regulation of protein function is a subject that has fascinated scientists for a long time. Although the implications and applications of selective ion binding are many, ranging from management of cell volume to proper electric signaling upon subsequent transport across cellular membranes to being integral part of new nano-materials, our understanding of the underlying complex thermodynamics of ion binding and subsequent permeation across cellular membranes is very limited. Recent progress in structural studies of secondary amino-acid transporters provides us with a unique opportunity to address molecular mechanism of the cation selectivity in the protein with available high-resolution crystal structure and confirmed Na+/K+ or K+/Na+ selectivity. Combined results of our studies on K-selective channels (KcsA and MthK), non-selective channels (NaK) and Na+-coupled secondary transporters will be presented. A combination of the free energy simulations with classical and polarizable force-fields as well as recently developed QM/MM FEP protocol was used for evaluation of different factors in the observed selectivity in protein sites. Atomistics simulations blazed the trail for the development of an analytical theory than allows quantification of ion selectivity within a reduced framework. In conclusion, I will present a theory to clarify the molecular determinants of ion selectivity in protein binding sites developed recently (Yu et al., PNAS 2011) that integrates our current knowledge on monovalent cation selectivity.

Functional Transfer of an Essential Aspartate for the Ion-binding Site in the Stator Proteins of the Bacterial Flagellar Motor

Rotation of the bacterial flagellar motor exploits the electrochemical potential of the coupling ion (H+ or Na+) as its energy source. In the marine bacterium Vibrio alginolyticus, the stator complex is composed of PomA and PomB, and conducts Na+ across the cytoplasmic membrane to generate rotation. The transmembrane (TM) region of PomB, which forms the Na+-conduction pathway together with TM3 and TM4 of PomA, has a highly conserved aspartate residue (Asp24) that is essential for flagellar rotation. This residue contributes to the Na+-binding site. However, it is not clear whether residues other than Asp24 are involved in binding the coupling ion. We examined the possibility that loss of the negative charge of Asp24 can be suppressed by introduction of negatively charged residues in TM3 or TM4 of PomA. The motility defect associated with the D24N substitution in PomB could be rescued only by a N194D substitution in PomA. This result suggests that there must be a negatively charged ion-binding pocket in the stator complex but that the presence of a negatively charged residue at position 24 of PomB is not essential. A tandemly fused PomA dimer containing the N194D mutation either in its N-terminal or C-terminal half with PomB-D24N was functional, suggesting that PomB-D24N can form an ion-binding pocket with either subunit of PomA dimer. The findings obtained in this study provide important clues to the mechanism of ion binding in the stator complex.

Copper-Induced Oligomerization of Peptides: A Model Study

In this work, copper-binding of the tetraglycine peptide (Gly-Gly-Gly-Gly) was studied by electrospray ionization mass spectrometry. Experiments were performed under alkaline conditions, in the presence of ethanolamine (pH 10.95). We observed that the presence of copper(II) ions induces the aggregation of the peptide and the formation of copper-bound complexes with higher molecular mass is favored, such as the oligomer complexes [3M+2Cu-3H](+) and [4M+3Cu-5H](+). At 1:1 peptide-copper(II) ion ratio, the singly charged [3M+2Cu-3H](+) oligomer complex is the base peak in the mass spectrum. Metal ion-induced oligomer-ization of neurotoxic peptides is well known in the literature; however, there are very few examples in which such oligomerization was directly observed by mass spectrometry. Our results show that application of short peptides can be useful to study the -mechanism of metal ion binding and metal ion-induced oligomerization of peptides.