Characterization of calciphorin by laser-excited europium luminescence

There is some question whether the calcium binding characteristics of calciphorin are due to contaminating phospholipids. To differentiate protein ion binding from ion binding by phospholipids or contaminating detergent, we describe here the use of Eu(III) as a metal-binding-site probe, and characterize the interaction of Eu(III) with calciphorin, cardiolipin, deoxycholate, and digitonin. The luminescence excitation pattern of Eu(III) bound to the calciphorin preparation clearly differentiates it from Eu(III) interactions with the possible contaminants. In addition, the effect of the luminescence decay constant of Eu(III) bound to calciphorin on the mole fraction of H₂O in a mixture of H₂O/²H₂O indicates that all except approximately 0.8 of the 9 to 10 water molecules coordinating Eu(III) in solution are stripped off upon binding to calciphorin. This also contrasts with the data for the possible contaminants.     

Studying metal ion binding properties of a three-way junction RNA by heteronuclear NMR

Self-splicing group II introns are highly structured RNA molecules, containing a characteristic secondary and catalytically active tertiary structure, which is formed only in the presence of Mg(II). Mg(II) initiates the first folding step governed by the κζ element within domain 1 (D1κζ). We recently solved the NMR structure of D1κζ derived from the mitochondrial group II intron ribozyme Sc.ai5γ and demonstrated that Mg(II) is essential for its stabilization. Here, we performed a detailed multinuclear NMR study of metal ion interactions with D1κζ, using Cd(II) and cobalt(III)hexammine to probe inner- and outer-sphere coordination of Mg(II) and thus to better characterize its binding sites. Accordingly, we mapped ¹H, ¹⁵N, ¹³C, and ³¹P spectral changes upon addition of different amounts of the metal ions. Our NMR data reveal a Cd(II)-assisted macrochelate formation at the 5′-end triphosphate, a preferential Cd(II) binding to guanines in a helical context, an electrostatic interaction in the ζ tetraloop receptor and various metal ion interactions in the GAAA tetraloop and κ element. These results together with our recently published data on Mg(II) interaction provide a much better understanding of Mg(II) binding to D1κζ, and reveal how intricate and complex metal ion interactions can be.     

The synthesis of (poly)hydroxycarboxylates Part IV. Eu(III) promoted O-alkylation of glycolate with maleate as studied on-line by luminescence

The O-alkylation of glycolate with maleate yielding carboxymethoxysuccinate (cmos) is a lanthanide(III) promoted reaction. It is demonstrated that the reaction can be studied on-line with the help of an optical fiber setup, monitoring the luminescence of the Eu(III) optical probe. During the reaction the ⁵D₀→F₀ transition shifts to lower wavenumbers and the average lifetime of the excited ⁵D₀ level of the Eu(III) ion increases, when substantial amounts of Eu(cmos)₂ are formed. The average number of OH oscillators in the first coordination sphere of the Eu(III) ion is decreased by two if one cmos per Eu(III) is formed. The concentration of cmos can be obtained by on-line measurements of the lifetime of the ⁵D₀ excited stare.     

A decadentate Gd(III)-coordinating paramagnetic cosolvent for protein relaxation enhancement measurement

Solvent paramagnetic relaxation enhancement (sPRE) arises from random collisions between paramagnetic cosolvent and protein of interest. The sPRE can be readily measured, affording protein structure information. However, lack of an inert cosolvent probe may yield sPRE values that are not consistent with protein structure. Here we synthesized a new sPRE probe, triethylenetetraamine hexaacetate trimethylamide gadolinium, or Gd(III)–TTHA–TMA. With a total of 10 coordination sites, this paramagnetic cosovlent eliminates an inner-sphere water molecule that can otherwise transfer relaxation to protein through exchange. With the metal ion centered, the new probe is largely spherical with a radius of 4.0 Å, permitting accurate back calculation of sPRE. The effectiveness Gd(III)–TTHA–TMA as a sPRE probe was demonstrated on three well-studied protein systems.     

Structure-Function Relationships in Iron and Manganese Superoxide Dismutases

Using the complete sequences for MnSOD from Thermus thermophilus and for FeSOD from E. coli, structural models for both oxidized enzymes have been refined, the Mn protein to an R of 0.186 for all data between 10.0 and 1.8 Å, and the Fe protein to an R of 0.22 for data between 10.0 and 2.5 A. The results of the refinements support the presence of a solvent as a fifth ligand to Mn(III) and Fe(III) and a coordination geometry that is close to trigonal bipyramidal. The putative substrate-entry channel is comprised of residues from both subunits of the dimer; several basic residues that are conserved may facilitate approach of O^?₂, while other conserved residues maintain interchain packing interactions. Analysis of the azide complex of Fe(III) dismutase suggests that during turnover O^?₂ binds to the metal at a sixth coordination site without displacing the solvent ligand. Because crystals reduced with dithionitc show no evidence for displacement of the protein ligands, the redox-linked proton acceptor (C. Bull and J.A. Fee (1985), Journol of the American Chemistry Society 107, 3295–3304) is unlikely to be one of the histidines which bind the metal ion. Structural, kinetic, titration, and spectroscopic data can be accommodated in a mechanistic scheme which accounts for the differential titration behaviour of the Fe(II1) and Fc(II) enzymes at neutral and high pH.     

Crystal structure of the trimeric N‐terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions

Centrin is a member of the EF‐hand superfamily of calcium‐binding proteins, a highly conserved eukaryotic protein that binds to Ca²⁺. Its self‐assembly plays a causative role in the fiber contraction that is associated with the cell division cycle and ciliogenesis. In this study, the crystal structure of N‐terminal domain of ciliate Euplotes octocarinatus centrin (N‐EoCen) was determined by using the selenomethionine single‐wavelength anomalous dispersion method. The protein molecules formed homotrimers. Every protomer had two putative Ca²⁺ ion‐binding sites I and II, protomer A, and C bound one Ca²⁺ ion, while protomer B bound two Ca²⁺ ions. A novel binding site III was observed and the Ca²⁺ ion was located at the center of the homotrimer. Several hydrogen bonds, electrostatic, and hydrophobic interactions between the protomers contributed to the formation of the oligomer. Structural studies provided insight into the foundation for centrin aggregation and the roles of calcium ions.     

Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

The small (87-residue) α-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an α-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and ?-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.     

Exploring the Ion Selectivity Properties of a Large Number of Simplified Binding Site Models

The ability to discriminate between different cations efficiently is essential for the proper physiological functioning of many membrane transport proteins. One obvious mechanism of ion selectivity is when a binding site is structurally constrained by the protein architecture and its geometry is precisely adapted to fit an ion of a given size. This mechanism is not effective in the case of flexible protein binding sites that are able to deform structurally or to adapt to a bound ion. In this study, the concept of nontrivial ion selectivity arising in a highly flexible protein binding site conceptually represented as a microdroplet of ligands confined to a small volume is explored. The environment imposed by the spatial confinement is a critical feature of the reduced models. A large number of reduced binding site models (1077) comprising typical ion-coordinating ligands (carbonyl, hydroxyl, carboxylate, water) are constructed and characterized for Na⁺/K⁺ and Ca²⁺/Ba²⁺ size selectivity using free energy perturbation molecular dynamics simulations. Free energies are highly correlated with the sum of ion-ligand and ligand-ligand mean interactions, but the relative balance of those two contributions is different for K⁺-selective and Na⁺-selective binding sites. The analysis indicates that both the number and the type of ligands are important factors in ion selectivity.     

Intraprotein electron transfer in a ruthenium-modified Tyr83-His plastocyanin mutant: evidence for strong electronic coupling

A site-directed mutant of spinach plastocyanin, Pc(Tyr83-His), has been modified by covalent attachment of a photoactive [Ru(bpy)₂(im)]²⁺ complex to the His83 residue. The residue is surface exposed and located about 10–12?Å from the copper ion at the entrance of a proposed natural electron transfer pathway from cytochrome f. Electron transfer within the Ru-Pc complex has been studied with time-resolved optical spectroscopy using two different approaches. In the first, the fully reduced [Cu(I), Ru(II)] protein was photoexcited and subsequently oxidized by an external quencher, forming the [Cu(I), Ru(III)] protein. This was followed by an electron transfer from reduced Cu(I) to Ru(III). In the second method, the initially oxidized Cu(II) ion acted as an internal quencher for excited Ru(II) and the photoinduced reduction of the Cu(II) ion was followed by a thermal recombination with the Ru(III) ion. The reoxidation of the Cu ion, which has an estimated driving force of 0.56?eV, occured with a rate constant k _et?=?(9.5±1.0)×10⁶?s^–1, observed with both methods. The results suggest a strong electronic coupling (H _DA>0.3?cm^–1) along the Ru-His(83)-Cys(84)-Cu pathway.     

New binuclear model compounds for the study of the 4f-4f exchange interaction

Two new homobinuclear 4f-4f complexes, [Ln^III(L)₃]₂, (Ln=Pr 1; Gd 2) have been synthesized (L⁻=2,6-diformyl-4-methylphenolato). The crystallographic investigation of 1 reveals centrosymmetric binuclear entities with the metallic centers connected by two of the anionic ligands. The praseodymium ions are bridged by the phenoxo oxygen atoms. One carbonyl group of a bridging molecule is coordinated to one Pr(III) ion, while the other one is coordinated to the second Pr(III) ion. The other four L⁻ ions act as terminal chelated ligands, through the phenoxo oxygen atom and one carbonyl group. The distance between the Pr(III) ions within the binuclear entity is 4.0711(4) Å. The supramolecular solid-state architecture is sustained by a system of π-π interactions. The cryomagnetic study of 2 reveals a very weak antiferromagnetic interaction between the Gd(III) ions (J=−0.053 cm⁻¹, ).     

Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca-induced membrane bridging

Annexin A2 (AnxA2) is a Ca²⁺- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca²⁺ binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca²⁺-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)₂, the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)₂ heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In “homotypic” membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in “homotypic” bridged membranes in the presence of Ca²⁺.     

Role of water-bridged interactions in metal ion coupled protein allostery

Allosteric communication between distant parts of proteins controls many cellular functions, in which metal ions are widely utilized as effectors to trigger the allosteric cascade. Due to the involvement of strong coordination interactions, the energy landscape dictating the metal ion binding is intrinsically rugged. How metal ions achieve fast binding by overcoming the landscape ruggedness and thereby efficiently mediate protein allostery is elusive. By performing molecular dynamics simulations for the Ca²⁺ binding mediated allostery of the calmodulin (CaM) domains, each containing two Ca²⁺ binding helix-loop-helix motifs (EF-hands), we revealed the key role of water-bridged interactions in Ca²⁺ binding and protein allostery. The bridging water molecules between Ca²⁺ and binding residue reduces the ruggedness of ligand exchange landscape by acting as a lubricant, facilitating the Ca²⁺ coupled protein allostery. Calcium-induced rotation of the helices in the EF-hands, with the hydrophobic core serving as the pivot, leads to exposure of hydrophobic sites for target binding. Intriguingly, despite being structurally similar, the response of the two symmetrically arranged EF-hands upon Ca²⁺ binding is asymmetric. Breakage of symmetry is needed for efficient allosteric communication between the EF-hands. The key roles that water molecules play in driving allosteric transitions are likely to be general in other metal ion mediated protein allostery.     

酿酒酵母细胞中内质网应激与未折叠蛋白反应的研究进展

内质网应激激活的未折叠蛋白反应(Unfolded protein response,UPR)途径在酿酒酵母和哺乳动物细胞中是非常保守的。内质网(Endoplasmic reticulum,ER)是蛋白质合成、折叠和修饰的细胞器,也是贮存钙的主要场所之一。酵母细胞内质网钙平衡与UPR的作用是相互的;两个MAPK途径——HOG途径和CWI途径都是细胞应答内质网应激压力时生存所必需的;重金属镉离子能够激活UPR途径,它通过激活钙离子通道Cch1/Mid1进入细胞影响钙离子的功能。本文结合最新研究进展对酿酒酵母细胞中的两个MAPK途径、镉离子和钙离子稳态与内质网应激激活的UPR途径之间相互关系进行综述。     

Synthesis, structure and magnetic properties of an oxo-bridged dinuclear iron(III) complex [(TPyA)FFeOFeF(TPyA)](BF4)2 · 0.5MeOH (TPyA = tris(2-pyridylmethyl)amine) containing the FFeOFeF linkage

The reaction of [Fe^II(H₂O)₆](BF₄)₂ with tris(2-pyridylmethyl)amine (TPyA) and triethylamine in methanol under aerobic conditions forms [(TPyA)FFe^IIIOFe^IIIF(TPyA)](BF₄)₂ · 0.5MeOH (1), in which each Fe(III) ion is coordinated to a TPyA and an F⁻ ion as well as an oxo ion (O²⁻) linking two Fe(III) ions. 1 has offset face-to-face π-π interactions between the dimers, and possesses a supramolecular network structure. The magnetic susceptibility of 1 can be fit with g = 2.0, J/k_B = − 153 K (106 cm⁻¹), and θ = − 0.3 K [H = − 2JS_a · S_b]. These indicate that very strong antiferromagnetic interactions occur via the oxo bridge within the Fe(III) dimer and weak antiferromagnetic interactions between the dimers.     

CHLOROTETRACYCLINE-ASSOCIATED FLUORESCENCE CHANGES DURING CALCIUM UPTAKE AND RELEASE BY RAT BRAIN SYNAPTOSOMES1

Abstract– The fluorescent divalent metal chelate-probe, chlorotetracycline (CTC), was used as a dynamic monitor of calcium association with rat brain snynaptosomes. The determined fluorescence excitation and emission maxima, 412 nm and 522 nm respectively, were used to monitor membrane-calcium interactions as a function of various parameters. Positive correlations were observed between increased or decreased fluorescence quantum yield and the uptake of both CTC and ⁴⁵Ca by synpatosomes. The divalent metal ionophore A23187 enhanced fluorescence as well as probe and ⁴⁵Ca uptake. Whereas, the polar chelator, EGTA, markedly reduced fluorescence, and the synaptosomal bound CTC and ⁴⁵Ca. The CTC fluorescence changes also demonstrated the saturable manner in which ⁴⁵Ca bound synaptosomes. At concentrations greater than 100μg/ml, CTC bound to the synaptosomes in a manner which quenched fluorescence at 522 nm. Also, CTC, at concentrations above 15 μg/ml, enhanced the uptake of ⁴⁵Ca. At CTC concentrations between 10 and 15 μg/ml the quenching and iono-phoretic properties of the probe were minimized without affecting the capability of using the probe to visualize calcium interactions with synaptosomal membranes. Also, at a low CTC concentration (12.5 μg/ml) the inhibition of calcium uptake by increasing monovalent ion concentrations was clearly demonstrated.     

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)₂(H₂O)₂⁺ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex.To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 µm magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.     

Possible existence of ion pairs at the mouths of ion channels

An electrostatic calculation suggests that when an ion is bound near the mouth of a channel penetrating a low-dielectric membrane, a counter ion may form an ion pair with this ion. The tendency towards ion-pair formation is remarkably enhanced at channel mouths by forces (image forces) arising from the charges induced on the boundaries between different dielectrics. The binding constant for the formation of ion-pairs of monovalent ions is estimated under the assumption that local interactions between the counter ion and the channel wall are negligibly small. It is of the order of 1–10 molal^?1 or more for the binding of a Cl^? (F^?) counter ion to an $\text{Na}{}^{\mathrm{+}}$ ($\text{Li}{}^{\mathrm{+}}$) ion if appropriate conditions are fulfilled. The binding constant depends on the position of the binding site, the dimensions and geometries of the channel and channel mouth, and the state of ion loading of the channel, as well as the ionic species. The present results also indicate that when cation (anion) channels have anionic (cationic) groups as integrant parts of their channel walls, interactions between these charged groups and permeant ions are markedly enhanced by the image forces.     

Ion–protein interactions of a potassium ion channel studied by attenuated total reflection Fourier transform infrared spectroscopy

An understanding of ion–protein interactions is key to a better understanding of the molecular mechanisms of proteins, such as enzymes, ion channels, and ion pumps. A potassium ion channel, KcsA, has been extensively studied in terms of ion selectivity. Alkali metal cations in the selectivity filter were visualized by X-ray crystallography. Infrared spectroscopy has an intrinsically higher structural sensitivity due to frequency changes in molecular vibrations interacting with different ions. In this review article, I attempt to summarize ion-exchange-induced differences in Fourier transform infrared spectroscopy, as applied to KcsA, to explain how this method can be utilized to study ion–protein interactions in the KcsA selectivity filter. A band at 1680 cm^?1 in the amide I region would be a marker band for the ion occupancy of K⁺, Rb⁺, and Cs⁺ in the filter. The band at 1627 cm^?1 observed in both Na⁺ and Li⁺ conditions suggests that the selectivity filter similarly interacts with these ions. In addition to the structural information, the results show that the titration of K⁺ ions provides quantitative information on the ion affinity of the selectivity filter.