Scanning conformational space with a library of stereo- and regiochemically diverse aminoglycoside derivatives: the discovery of new ligands for RNA hairpin sequences

A library of stereo- and regiochemically diverse aminoglycoside derivatives was screened at 1 microM using surface plasmon resonance (SPR) against RNA hairpin models of the bacterial A-site, and the HIV viral TAR and RRE sequences. In order to double the stereochemical diversity of the library, the compounds were screened against both enantiomers of each of these sequences. Remarkably, this initial screen suggested that the same four aminoglycoside derivatives bound most tightly to all three of the RNAs, suggesting that these compounds were good RNA binders which, nonetheless, discriminated poorly between the RNA sequences. The interactions between selected isomeric aminoglycoside derivatives and the RNA hairpins were then studied in more detail using an SPR assay. Three isomeric tight-binding aminoglycoside derivatives, which had been identified from the initial screen, were found to bind more tightly to the RNA hairpins (with K(D) values in the range 0.23 to 4.7 microM) than a fourth isomeric derivative (which had K(D) values in the range 6.0 to 30 microM). The magnitude of the tightest RNA-aminoglycoside interactions stemmed, in large part, from remarkably slow dissociation of the aminoglycosides from the RNA targets. The three tight-binding aminoglycoside derivatives were found, however, to discriminate rather poorly between alternative RNA sequences with, at best, around a twenty-fold difference in affinity for alternative RNA hairpin sequences. Within the aminoglycoside derivative library studied, high affinity for an RNA target was not accompanied by good discrimination between alternative RNA sequences.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) and water coordination on the structure of glycine and zwitterionic glycine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. Here, the effect of metal ions and water on the structure of glycine is examined. The effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) and water on structures of Gly.Mn+(H2O)m and GlyZwitt.Mn+(H2O)m (m = 0, 2, 5) complexes have been determined theoretically by employing the hybrid B3LYP exchange-correlation functional and using extended basis sets. Selected calculations were carried out also by means of CBS-QB3 model chemistry. The interaction enthalpies, entropies, and Gibbs energies of eight complexes Gly.Mn+ (Mn+ = Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) were determined at the B3LYP density functional level of theory. The computed Gibbs energies DeltaG degrees are negative and span a rather broad energy interval (from -90 to -1100 kJ mol(-1)), meaning that the ions studied form strong complexes. The largest interaction Gibbs energy (-1076 kJ mol(-1)) was computed for the NiGly2+ complex. Calculations of the molecular structure and relative stability of the Gly.Mn+(H2O)m and GlyZwitt.Mn+(H2O)m (Mn+ = Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+; m = 0, 2, and 5) systems indicate that in the complexes with monovalent metal cations the most stable species are the NO coordinated metal cations in non-zwitterionic glycine. Divalent cations Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+ prefer coordination via the OO bifurcated bonds of the zwitterionic glycine. Stepwise addition of two and five water molecules leads to considerable changes in the relative stability of the hydrated species. Addition of two water molecules at the metal ion in both Gly.Mn+ and GlyZwitt.Mn+ complexes reduces the relative stability of metallic complexes of glycine. For Mn+ = Li+ or Na+, the addition of five water molecules does not change the relative order of stability. In the Gly.K+ complex, the solvation shell of water molecules around K+ ion has, because of the larger size of the potassium cation, a different structure with a reduced number of hydrogen-bonded contacts. This results in a net preference (by 10.3 kJ mol(-1)) of the GlyZwitt.K+H2O5 system. Addition of five water molecules to the glycine complexes containing divalent cations Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+ results in a net preference for non-zwitterionic glycine species. The computed relative Gibbs energies are quite high (-10 to -38 kJ mol(-1)), and the NO coordination is preferred in the Gly.Mn+(H2O)5 (Mn+ = Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) complexes over the OO coordination.     

Probing conformational variations at the ATPase site of the RNA helicase DbpA by high-field electron-nuclear double resonance spectroscopy

The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein's ATPase site in solution. Specifically, we substituted the essential Mg(2+) cofactor in the ATPase active site for paramagnetic Mn(2+) and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn(2+) interactions with the nucleotide phosphates through the (31)P hyperfine couplings and the coordination by protein residues through (13)C hyperfine coupling from (13)C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the (13)C and the (31)P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn(2+) ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn(2+) cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the protein's ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn(2+) ion. Finally, a competitive Mn(2+) binding site was found for single-stranded RNA construct.     

A joint experimental and theoretical study of cation-pi interactions: multiple-decker sandwich complexes of ferrocene with alkali metal ions (Li+, Na+, K+, Rb+, Cs+)

A systematic study of cation-pi interactions between alkali metal ions and the cyclopentadienyl ring of ferrocene is presented. The alkali metal (Li+, Na+, K+, Rb+, Cs+) salts of the ditopic mono(pyrazol-1-yl)borate ligand [1,1'-fc(BMe2pz)2]2- crystallize from dimethoxyethane as multiple-decker sandwich complexes with the M+ ions bound to the pi faces of the ferrocene cyclopentadienyl rings in an eta5 manner (fc = (C5H4)2Fe; pz = pyrazolyl). X-ray crystallography of the lithium complex reveals discrete trimetallic entities with each lithium ion being coordinated by only one cyclopentadienyl ring. The sodium salt forms polyanionic zigzag chains where each Na+ ion bridges the cyclopentadienyl rings of two ferrocene moieties. Linear columns [-CpR-Fe-CpR-M+-CpR-Fe-CpR-M+-](infinity) (R = [-BMe2pz]-) are established by the K+, Rb+, and Cs+ derivatives in the solid state. According to DFT calculations, the binding enthalpies of M+-eta5(ferrocene) model complexes are about 20% higher as compared to the corresponding M+-eta6(benzene) aggregates when M+ = Li+ or Na+. For K+ and Rb+, the degree of cation-pi interaction with both aromatics is about the same. The binding sequence along the M+-eta5(ferrocene) series follows a classical electrostatic trend with the smaller ions being more tightly bound.     

Voltammetric study of the ion-exchange binding of non-electroactive metal cations to DNA-modified surfaces

We describe a simple electrochemical protocol for studying the ion-exchange binding of non-electroactive ions, specifically mono- and divalent metal cations of biological relevance (Mg(2+), Ca(2+), and K(+)), to DNA-modified surfaces. After incubation in a dilute solution of multiply charged transition metal complex (5.0 microM [Ru(NH(3))(6)]Cl(3)), gold electrodes modified with thiolate-DNA monolayers respond to the presence of these non-electroactive metal cations by producing significant changes in the cyclic voltammograms (i.e., decrease of the integrated charge and shift of formal potential) of the surface-bound redox complex ([Ru(NH(3))(6)](3+)). The divalent cations (particularly Mg(2+)) can be detected at very low concentrations (<10 microM), while the on-set value for K(+) is substantially higher (50 mM). The equilibrium binding constants for Mg(2+) and Ca(2+) to DNA-modified surfaces were calculated.     

The liquidlike ordering of lipid A-diphosphate colloidal crystals: the influence of Ca2+, Mg2+, Na+, and K+ on the ordering of colloidal suspensions of lipid A-diphosphate in aqueous solutions

A comprehensive study was performed on electrostatically stabilized aqueous dispersion of lipid A-diphosphate in the presence of bound Ca2+, Mg2+, K+, and Na+ ions at low ionic strength (0.10-10.0-mM NaCl, 25 degrees C) over a range of volume fraction of 1.0 x 10(-4)< or =phi< or =4.95 x 10(-4). These suspensions were characterized by light scattering (LS), quasielastic light scattering, small-angle x-ray scattering, transmission electron microscopy, scanning electron microscopy, conductivity measurements, and acid-base titrations. LS and electron microscopy yielded similar values for particle sizes, particle size distributions, and polydispersity. The measured static structure factor, S(Q), of lipid A-diphosphate was seen to be heavily dependent on the nature and concentration of the counterions, e.g., Ca2+ at 5.0 nM, Mg2+ at 15.0 microM, and K+ at 100.0 microM (25 degrees C). The magnitude and position of the S(Q) peaks depend not only on the divalent ion concentration (Ca2+ and Mg2+) but also on the order of addition of the counterions to the lipid A-diphosphate suspension in the presence of 0.1-microM NaCl. Significant changes in the rms radii of gyration (R2G) 1/2 of the lipid A-diphosphate particles were observed in the presence of Ca2+ (24.8+/-0.8 nm), Mg2+ (28.5+/-0.7 nm), and K+ (25.2+/-0.6 nm), whereas the Na+ salt (29.1+/-0.8 nm) has a value similar to the one found for the de-ionized lipid A-diphosphate suspensions (29.2+/-0.8 nm). Effective particle charges were determined by fits of the integral equation calculations of the polydisperse static structure factor, S(Q), to the light-scattering data and they were found to be in the range of Z*=700-750 for the lipid A-diphosphate salts under investigation. The light-scattering data indicated that only a small fraction of the ionizable surface sites (phosphate) of the lipid A-diphosphate was partly dissociated (approximately 30%). It was also discovered that a given amount of Ca2+ (1.0-5.0 nM) or K+ (100 microM) influenced the structure much more than Na+ (0.1-10.0-mM NaCl) or Mg2+ (50 microM). By comparing the heights and positions of the structure factor peaks S(Q) for lipid A-diphosphate-Na+ and lipid A-diphosphate-Ca2+, it was concluded that the structure factor does not depend simply on ionic strength but more importantly on the internal structural arrangements of the lipid A-diphosphate assembly in the presence of the bound cations. The liquidlike interactions revealed a considerable degree of ordering in solution accounting for the primary S(Q) peak and also the secondary minimum at large particle separation. The ordering of lipid A-diphosphate-Ca2+ colloidal crystals in suspension showed six to seven discrete diffraction peaks and revealed a face-centered-cubic (fcc) lattice type (a=56.3 nm) at a volume fraction of 3.2 x 10(-4)< or =phi< or =3.9 x 10(-4). The K+ salt also exhibited a fcc lattice (a=55.92 nm) at the same volume fractions, but reveals a different peak intensity distribution, as seen for the lipid A-diphosphate-Ca2+ salt. However, the Mg2+ and the Na+ salts of lipid A-diphosphate showed body-centered-cubic (bcc) lattices with a=45.50 nm and a=41.50 nm, respectively (3.2 x 10(-4)< or =phi< or =3.9 x 10(-4)), displaying the same intensity distribution with the exception of the (220) diffraction peaks, which differ in intensity for both salts of lipid A-diphosphate.     

Targeted cleavage of HIV RRE RNA by Rev-coupled transition metal chelates

A series of compounds that target reactive metal chelates to the HIV-1 Rev response element (RRE) mRNA have been synthesized. Dissociation constants and chemical reactivity toward HIV RRE RNA have been determined and evaluated in terms of reduction potential, coordination unsaturation, and overall charge associated with the metal-chelate-Rev complex. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine side chain of a Rev-derived peptide by either EDC/NHS or isothiocyanate coupling. The resulting chelate-Rev (EDTA-Rev, DTPA-Rev, NTA-Rev, and DOTA-Rev) conjugates were used to form coordination complexes with Fe(2+), Co(2+), Ni(2+), and Cu(2+) such that the arginine-rich Rev peptide could mediate localization of the metal chelates to the Rev peptide's high-affinity mRNA binding partner, RRE stem loop IIB. Metal complexes of the extended peptides GGH-Rev and KGHK-Rev, which also contain N-terminal peptidic chelators (ATCUN motifs), were studied for comparison. A fluorescence titration assay revealed high-affinity RRE RNA binding by all 22 metal-chelate-Rev species, with K(D) values ranging from ~0.2 to 16 nM, indicating little to no loss of RNA affinity due to the coupling of the metal chelates to the Rev peptide. Dissociation constants for binding at a previously unobserved low-affinity site are also reported. Rates of RNA modification by each metal-chelate-Rev species were determined and varied from ~0.28 to 4.9 nM/min but were optimal for Cu(2+)-NTA-Rev. Metal-chelate reduction potentials were determined and varied from -228 to +1111 mV vs NHE under similar solution conditions, allowing direct comparison of reactivity with redox thermodynamics. Optimal activity was observed when the reduction potential for the metal center was poised between those of the two principal co-reagents for metal-promoted formation of reactive oxygen species: E°(ascorbate/ascorbyl radical) = -66 mV and E°(H(2)O(2)/hydroxyl radical) = 380 mV. Given the variety of oxidative activities of these metal complexes and their high-affinity binding to the targeted RRE mRNA following coupling to the Rev peptide, this class of metal-chelate-Rev derivatives constitutes a promising step toward development of multiple-turnover reagents for selective eradication of HIV-1 RRE mRNA.     

A new emission band of Eu2+ and its efficient energy transfer to Mn2+ in Sr2Mg3P4O15:Mn2+, Eu2+

Eu(2+) singly and Eu(2+), Mn(2+) co-doped Sr(2)Mg(3)P(4)O(15) exhibit not only the well known blue emission band of Eu(2+) peaking at 448 nm but also a new band at 399 nm in violet. They are attributed to Eu(2+) on different Sr(2+) sites. The Eu(2+) for the violet band can transfer energy to the red emitting Mn(2+) more efficiently than Eu(2+) for the blue band. The new Eu(2+) band could enable Sr(2)Mg(3)P(4)O(15):Mn(2+), Eu(2+) to be a promising phosphor for enriching the red component of white LEDs.     

Ionophoric properties of atropine: complexation and transport of Na+, K+, Mg2+ and Ca2+ ions across a liquid membrane

The activity of atropine on the complexation and transport of Na(+), K(+), Mg(2+) and Ca(2+) ions across a liquid membrane was investigated using a spectrophotometric method. Atropine is a natural drug that blocks muscarinic receptors. It is a competitive antagonist of the action of acetylcholine and other muscarinic agonists. Atropine is shown to extract Na(+), K(+), Mg(2+) and Ca(2+) ions from an aqueous phase into an organic one with a preference for Ca(2+) ions. According to a kinetic study, divalent cations (Mg(2+) and Ca(2+)) are more rapidly transported than monovalent ones (Na(+) and K(+)). In both complexation and transport, the flux of the ions increases with the increase of atropine concentration. Atropine might act on the membrane permeability; its complexation and ionophoric properties shed new lights on its therapeutic properties.     

Comprehensive study on the solvation of mono- and divalent metal cations: Li+, Na+, K+, Be2+, Mg2+ and Ca2+

Hydration of mono- and divalent metal ions (Li(+), Na(+), K(+), Be(2+), Mg(2+) and Ca(2+)) has been studied using the DFT (B3LYP), second-order M?ller-Plesset (MP2) and CCSD(T) perturbation theory as well as the G3 quantum chemical methods. Double-zeta and triple-zeta basis sets containing both (multiple) polarization and diffuse functions were applied. Total and sequential binding energies are evaluated for all metal-water clusters containing 1-6 water molecules. Total binding energies predicted at lower levels of theory are compared with those from the high level G3 calculations, whereas the sequential binding energies are compared with available experimental values. An increase in the quality of the basis set from double-zeta to triple-zeta has a significant effect on the sequential binding energies, irrespective of the geometries used. Within the same group (I or II), the sequential binding energy predictions at the MP2 and B3LYP vary appreciably. We noticed that, for each addition of a water molecule, the change of the M-O distance in metal-water clusters is higher at the B3LYP than at the MP2 level. The charge of the metal ion decreases monotonically as the number of water molecules increase in the complex.     

Evidence for a thermodynamically distinct Mg2+ ion associated with formation of an RNA tertiary structure

A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg(2+)-RNA chelates could be responsible for a significant fraction of the Mg(2+)-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg(2+) chelation is uncoupled from RNA folding and from unfavorable interactions with Mg(2+) ions in other environments. In a 58mer rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg(2+)- and protein-stabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg(2+) concentration, and is partially resolved from the accumulation of Mg(2+) in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg(2+)-RNA interaction free energies over the range of accessed Mg(2+) concentrations. At 0.1 mM Mg(2+) and 60 mM K(+), specific site binding contributes ～-3 kcal/mol of the total Mg(2+) interaction free energy of ～-13 kcal/mol from all sources; at higher Mg(2+) concentrations the site-binding contribution becomes a smaller proportion of the total (-4.5 vs -33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but will provide only a fraction of the total free energy of Mg(2+)-RNA interactions.     

Fluorescent HIV-1 Dimerization Initiation Site: design, properties, and use for ligand discovery

The HIV-1 Dimerization Initiation Site (DIS) is an intriguing, yet underutilized, viral RNA target for potential antiretroviral therapy. To study the recognition features of this target and to provide a quantitative, rapid, and real-time tool for the discovery of new binders, a fluorescence-based assay has been constructed. It relies on strategic incorporation of 2-aminopurine, an isosteric fluorescent adenosine analogue, into short hairpin RNA constructs. These oligomers self-associate to form a kissing loop that thermally rearranges into a more stable extended duplex, thereby mimicking the association and structural features of the native RNA sequence. We demonstrate the ability of two fluorescent DIS constructs, DIS272(2AP) and DIS273(2AP), to report the binding of known DIS binders via changes in their emission intensity. Binding of aminoglycosides such as paromomycin to DIS272(2AP) results in significant fluorescence enhancement, while ligand binding to DIS273(2AP) results in fluorescence quenching. These observations are rationalized by comparison to the sequence-analogous bacterial A-site, where the relative emission of the fluorescent probe is dependent on the placement of the flexible purine residues inside or outside the helical domain. Analysis of binding isotherms generated using DIS272(2AP) yields submicromolar EC50 values for paromomycin (0.5 +/- 0.2 microM) and neomycin B (0.6 +/- 0.2 microM). Other neomycin-family aminoglycosides are less potent binders with neamine, the core pharmacophore, displaying the lowest affinity of 21 +/- 1 microM. Screening of additional aminoglycosides and their derivatives led to the discovery of new, previously unreported, aminoglycoside binders of the HIV DIS RNA, among them butirosin A (5.5 +/- 0.6 microM) and apramycin (7.6 +/- 1.0 microM). A conformationally constrained neomycin B analogue displays a rather high affinity to the DIS (1.9 +/- 0.2 microM). Among a series of nucleobase aminoglycoside conjugates, only the uracil derivatives display a measurable affinity using this assay with EC50 values in the 2 microM range. In addition, similarity between the solution behavior of HIV-1 DIS and the bacterial decoding A-site has been observed, particularly with respect to the intra- and extra-helical residence of the conformationally flexible A residues within the bulge. Taken together, the observations reported here shed light on the solution behavior of this important RNA target and are likely to facilitate the design of new DIS selective ligands as potential antiretroviral agents.     

Binding of Ca2+, Mg2+, and heparin by human serum amyloid P component in affinity capillary electrophoresis

Human serum amyloid P component (SAP) is a glycoprotein circulating in the blood and found in association with all types of amyloid (malfolded potein aggregates) examined so far. Despite uncertainties regarding the precise function of SAP in vivo, the lectin-like properties of this Ca(2+)-activated protein with affinity for anionic saccharides and malfolded proteins are well known. The propensity to form homomeric penta- or decamers in solution and the selfaggregation in the presence of Ca(2+) as well as the tendency of SAP to attach to uncoated fused silica have precluded the analysis of SAP by microelectrophoretic methods. We now work out conditions to characterize the binding of Ca(2+) and Mg(2+) and the binding of heparin to SAP in the presence of divalent metal ions by ACE. The results show a strong binding of heparin (sub-muM apparent dissociation constants) even in the abscence of Ca(2+) at low ionic strength, pH 8.2. Also, a selective interaction with Ca(2+) compared with Mg(2+) is demonstrated. The approach will further the use of microelectrophoretic methods to examine the interactions of SAP with ligands of putative pathophysiological relevance such as lipopolysaccharides and misfolded proteins.     

Use of phosphorus ligand NMR probes to investigate electronic and second-sphere solvent effects in ligand substitution reactions at manganese(II) and manganese(III)

Manganese/ligand association dynamics were studied using a series of structurally related anionic phosphorus ester ligand probes [CH(3)OP(O)(X)(Y)(-), where X = CH(3)O, CH(3)CH(2), or H and Y = O, S, or BH(3)]. Reactions of the probe ions with Mn(H(2)O)(6)(2+) and a manganese(III) porphyrin (Mn(III)TMPyP(5+)) were studied in aqueous solution by paramagnetic (31)P NMR line-broadening techniques. A satisfactory linear free energy relationship for reactions of the probe ions with Mn(H(2)O)(6)(2+) and Mn(III)TMPyP(5+) required consideration of both the basicity and solvent affinity of the probe ligands: log(k(app)) = log(k(0)) + alpha pK(a) + beta log(K(ext)), where k(0), alpha, and beta are metal complex dependent parameters and pK(a) and K(ext) represent the measured Bronsted acidity and water/n-butanol extraction constant for the probe anions, respectively. Reactions of Mn(H(2)O)(6)(2+) were relatively insensitive to changes in ligand basicity (alpha = -0.04) and favored the more hydrophilic anions (beta = -0.54). These observations are consistent with a dissociative ligand exchange mechanism wherein the outer-sphere complex is stabilized by hydrogen bonding between Mn(H(2)O)(6)(2+) and the incoming ligand. In contrast, reactions with Mn(III)TMPyP(5+) are accelerated by decreases in both the basicity (alpha = -0.43) and the hydrophilicity (beta = +0.97) of the probe. We conclude that reactions of Mn(III)TMPyP(5+) are also dissociative but that the aromatic groups of the porphyrin provide a hydrophobic environment surrounding the ligand binding site in Mn(III)TMPyP(5+). Thus, the probe/water solvent interactions must be significantly weakened in order to form the outer-sphere complex that leads to ligand substitution. This work demonstrates the utility of phosphorus relaxation enhancement (PhoRE) techniques for characterizing the second coordination sphere environment of metal complexes leading to ligation and will allow comparison of the second coordination spheres of Mn(H(2)O)(6)(2+) and Mn(III)TMPyP(5+) to those of other metal complexes.     

Conformational constraint as a means for understanding RNA-aminoglycoside specificity

The lack of high RNA target selectivity displayed by aminoglycoside antibiotics results from both their electrostatically driven binding mode and their conformational adaptability. The inherent flexibility around their glycosidic bonds allows them to easily assume a variety of conformations, permitting them to structurally adapt to diverse RNA targets. This structural promiscuity results in the formation of aminoglycoside complexes with diverse RNA targets in which the antibiotics assume distinct conformations. Such differences suggest that covalently linking individual rings in an aminoglycoside could reduce its available conformations, thereby altering target selectivity. To explore this possibility, conformationally constrained neomycin and paromomycin analogues designed to mimic the A-site bound aminoglycoside structure have been synthesized and their affinities to the TAR and A-site, two therapeutically relevant RNA targets, have been evaluated. As per design, this constraint has minimal deleterious effect on binding to the A-site. Surprisingly, however, preorganizing these neomycin-class antibiotics into a TAR-disfavored structure has no deleterious effect on binding to this HIV-1 RNA sequence. We rationalize these observations by suggesting that the A-site and HIV TAR possess inherently different selectivities toward aminoglycosides. The inherent plasticity of the TAR RNA, coupled to the remaining flexibility within the conformationally constrained analogues, makes this RNA site an accommodating target for such polycationic ligands. In contrast, the deeply encapsulating A-site is a more discriminating RNA target. These observations suggest that future design of novel target selective RNA-based therapeutics will have to consider the inherent "structural" selectivity of the RNA target and not only the selectivity patterns displayed by the low molecular weight ligands.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) and water coordination on the structure and properties of L-arginine and zwitterionic L-arginine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. The effect of metal ions and water on the structure of L-arginine is examined. The effects of metal ions (Li(+), Na(+), K(+), Mg(2+), Ca(2+), Ni(2+), Cu(2+), and Zn(2+)) and water on structures of Arg x M(H2O)m , m = 0, 1 complexes have been determined theoretically by employing the density functional theories (DFT) and using extended basis sets. Of the three stable complexes investigated, the relative stability of the gas-phase complexes computed with DFT methods (with the exception of K(+) systems) suggests metallic complexes of the neutral L-arginine to be the most stable species. The calculations of monohydrated systems show that even one water molecule has a profound effect on the relative stability of individual complexes. Proton dissociation enthalpies and Gibbs energies of arginine in the presence of the metal cations Li(+), Na(+), K(+), Mg(2+), Ca(2+), Ni(2+), Cu(2+), and Zn(2+) were also computed. Its gas-phase acidity considerably increases upon chelation. Of the Lewis acids investigated, the strongest affinity to arginine is exhibited by the Cu(2+) cation. The computed Gibbs energies DeltaG(o) are negative, span a rather broad energy interval (from -150 to -1500 kJ/mol), and are appreciably lowered upon hydration.     

Coordination environment of a site-bound metal ion in the hammerhead ribozyme determined by 15N and 2H ESEEM spectroscopy

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a K(d,app) < 10 microM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin-echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands.     

Properties of (Mg2 + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: influence of Mg2+, Ca2+, ATP, and protein activator.

Erythrocyte membranes prepared by three different procedures showed (Mg2+ + Ca2+)-ATPase activities differing in specific activity and in affinity for Ca2+. The (Mg2+ + Ca2+)-ATPase activity of the three preparations was stimulated to different extents by a Ca2+-dependent protein activator isolated from hemolysates. The Ca2+ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2-200 microM or lowering the Mg:ATP ratio to less than one shifted the (Mg2+ + Ca2+)-ATPase activity in stepwise hemolysis membranes from mixed "high" and "low" affinity to a single high Ca2+ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strength, or membranes prepared by the EDTA (1-10 mM) procedure, did not undergo the shift in the Ca2+ affinity with changes in ATP and MgCl2 concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg2+ + Ca2+)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Magnesium fluctuations modulate RNA dynamics in the SAM-I riboswitch

Experiments demonstrate that Mg(2+) is crucial for structure and function of RNA systems, yet the detailed molecular mechanism of Mg(2+) action on RNA is not well understood. We investigate the interplay between RNA and Mg(2+) at atomic resolution through ten 2-μs explicit solvent molecular dynamics simulations of the SAM-I riboswitch with varying ion concentrations. The structure, including three stemloops, is very stable on this time scale. Simulations reveal that outer-sphere coordinated Mg(2+) ions fluctuate on the same time scale as the RNA, and that their dynamics couple. Locally, Mg(2+) association affects RNA conformation through tertiary bridging interactions; globally, increasing Mg(2+) concentration slows RNA fluctuations. Outer-sphere Mg(2+) ions responsible for these effects account for 80% of Mg(2+) in our simulations. These ions are transiently bound to the RNA, maintaining interactions, but shuttled from site to site. Outer-sphere Mg(2+) are separated from the RNA by a single hydration shell, occupying a thin layer 3-5 ? from the RNA. Distribution functions reveal that outer-sphere Mg(2+) are positioned by electronegative atoms, hydration layers, and a preference for the major groove. Diffusion analysis suggests transient outer-sphere Mg(2+) dynamics are glassy. Since outer-sphere Mg(2+) ions account for most of the Mg(2+) in our simulations, these ions may change the paradigm of Mg(2+)-RNA interactions. Rather than a few inner-sphere ions anchoring the RNA structure surrounded by a continuum of diffuse ions, we observe a layer of outer-sphere coordinated Mg(2+) that is transiently bound but strongly coupled to the RNA.