23Na NMR investigation of the interaction between DNA and divalent metallic cations

The aim of this study is to follow the thermodynamic behaviour of Na⁺ ions, acting as natural counterions of DNA, in the presence of divalent metal ions, by using the²³Na NMR technique. With the help of the²³Na entropy of fluctuations concept introduced by Lenk, we propose the following decreasing sequence: Mg⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, and Cu⁺⁺, for the magnitude of divalent metal ions interactions with DNA phosphate sites.     

The interaction of polyamines with DNA: a 23Na NMR study.

The interaction between a variety of polyamines, both naturally occurring and synthetic, and calf thymus DNA has been studied using 23Na NMR. The relaxation behaviour of 23Na reflects the extent of interaction of Na+ with DNA phosphate groups and therefore the extent of charge neutralisation of DNA phosphate groups (P) by polyamine amino and imino groups (N) in solutions of DNa, polyamine and Na+. The studies reveal that whereas spermine and spermidine are capable of expelling nearly all of the Na+ ions from DNA at N/P approximately 1, diamines such as putrescine and homologues of spermine and spermidine are capable of neutralising only roughly 50% of DNA phosphates. The results provide a challenge to current models of DNA-polyamine interactions.     

Direct interaction between SNAP-23 and L-type Ca2+ channel

During secretion, membrane-bound secretory vesicles dock and fuse at the base of porosomes in the cell plasma membrane. Among other proteins, the porosome is composed of SNAREs and Ca2+-channels. Ca2+-channels and SNAREs have been implicated in cell secretion. Several immunoprecipitation and binding studies suggest the physical interaction of the t-SNARE proteins, Syntaxin-1 and SNAP-25 with various Ca2+-channels. In this study, using yeast two-hybrid and immunoanalysis, we demonstrate for the first time, direct interaction of SNAP-23 and a L-type Ca2+-channel at the plasma membrane in pancreas.     

Double-quantum-filtered 23Na NMR study of intracellular sodium in the perfused liver.

We acquired double-quantum-filtered 23Na NMR spectra from perfused liver, using a range of tau values from 0.2 to 24 ms, where tau is the separation between the first and second pi/2 pulses in the radio-frequency pulse sequence. For each tau value we compared the amplitude of the double-quantum-filtered 23Na NMR signal acquired from intracellular sodium ions when the liver was perfused with buffer containing the "shift reagent" Dy(PPP)2 to the amplitude of the total double-quantum-filtered 23Na NMR signal acquired when the liver was perfused with buffer containing no Dy(PPP)2. For tau < or = 4 ms, the average ratio of the two amplitudes was 0.98 +/- 0.03 (mean +/- SEM). For tau > or = 8 ms, the average ratio was significantly less than 1. These results demonstrate that double-quantum-filtered 23Na NMR signals acquired from perfused liver using short tau values arise almost exclusively from intracellular sodium ions, but double-quantum-filtered 23Na NMR signals acquired from perfused liver using long tau values contain contributions from both intracellular and extracellular sodium ions. This conclusion suggests that multiple-quantum-filtered 23Na NMR spectroscopy will be useful in studying intracellular sodium levels in the perfused liver, and possibly in the intact liver in vivo.     

23Na NMR study of intracellular sodium ions in Dictyostelium discoideum amoeba

The intracellular sodium concentration in the amoebae from the slime mold Dictyostelium discoideum has been studied using 23Na NMR. The 23Na resonances from intracellular and extracellular compartments could be observed separately in the presence of the anionic shift reagent Dy(PPPi)7-2 which does not enter into the amoebae and thus selectively affects Na+ in the extracellular space. 31P NMR was used to control the absence of cellular toxicity of the shift reagent. The intracellular Na+ content was calculated by comparison of the intensities of the two distinct peaks arising from the intra- and extracellular spaces. It remained low (0.6 to 3 mM) in the presence of external Na+ (20 to 70 mM), and a large Na+ gradient (20- to 40-fold) was maintained. A rapid reloading of cells previously depleted of Na+ was readily measured by 23Na NMR. Nystatin, an antibiotic known to perturb the ion permeability of membranes, increased the intracellular Na+ concentration. The time dependence of the 23Na and 31P NMR spectra showed a rapid degradation of Dy(PPPi)7-2 which may be catalyzed by an acid phosphatase.     

23Na NMR study of DNA thermal transconformation in presence of cysteamine radioprotector

DNA thermal transconformation is studied in absence and in presence of the cysteamine radioprotector, by observing the delta nu 1/2 variation of 23Na NMR peaks. The sodium state (Free or Bound) is discussed with the help of a two states model with RF and RB relaxation rates. The delta nu 1/2 behaviour during the DNA transconformation shows clearly the electrostatic interaction with cysteamine which is accompanied by an Na+ ejection out of phosphate sites. The temperature dependence of delta nu 1/2 in all cases leads to the conclusion that RBc (the average relaxation rate of sodium nuclei that remain bound in the coil state of DNA) tends to zero.     

23Na NMR study of the effect of organic osmolytes on DNA counterion atmosphere.

The effect of different organic osmolytes on the DNA counterion condensation layer has been investigated by 23Na NMR relaxation measurements. The zwitterionic compounds glycine, beta-alanine, 4-aminobutyric acid, and 6-aminocaproic acid have shown an increasing capacity to decrease the amount of sodium ions in the vicinity of the macromolecule. The experimental data have been correlated with the dielectric constant increase in their corresponding solutions and have been compared with the prediction of counterion condensation theory. Polyols (sorbitol and mannitol) did not display the same effect. These compounds largely increase the relaxation rate of sodium ions in the proximity of DNA, unlike the zwitterionic compounds. This probably results from a perturbation of the water dynamic around the macromolecule, of the primary or secondary hydration shell of the sodium nuclei involved, or both.     

Specificity of Na+ binding to phosphatidylserine vesicles from a 23Na NMR relaxation rate study.

23Na NMR relaxation rate measurements show that Na+ binds specifically to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4--1.2 M-1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration.     

48 NMR study of the interaction between G-quadruplexes and small molecules

Guanine-rich oligonucleotides are able to adopt secondary DNA structures, known as G-quadruplexes. Such G-rich sequences are found in human telomeres, promoter regions of oncogenes, 5′ untranslated regions (UTRs) of mRNAs and human intronic sequences. Studies have shown that small molecules can induce anti-cancer effect through stabilizing or promoting G-quadruplex formation. In order to design and develop a potent drug, structural details on the interaction between small molecules and G-quadruplexes are invaluable. In this study, we seek to understand the structural determinants involved in the interaction between G-quadruplexes and small molecules. NMR spectroscopy is employed to resolve the structures of two intramolecular G-quadruplexes bound to small molecules. These resolved complexes allow us to structurally design new potent drugs for their application in anti-cancer therapy.     

23Na NMR maps of head sized phantoms and a low resolution 23Na map of the live human head

An NMR system capable of obtaining 23Na NMR signals and scans from large objects containing biological concentrations of sodium in a 411 gauss magnetic field in less than one hour was developed. Scans were carried out on 6"-8" diameter phantoms containing 150 mM NaCl and the first low resolution 23Na NMR map of a live human head was obtained.     

Primary mutations in calmodulin prevent activation of the Ca(++)-dependent Na+ channel in Paramecium.

Paramecium tetraurelia behavioral mutant cam12 displays a "fast-2" behavioral phenotype: it fails to respond to Na+ stimuli. Electrophysiologically, it lacks a Ca(++)-dependent Na+ current. Genetics and DNA sequencing showed the primary defect of cam12 to be in the calmodulin gene (Kink et al., 1990). To correlate calmodulin structure and function in Paramecium, we elucidated the primary structure of cam12 calmodulin. Peptide sequencing confirmed the two point mutations predicted by the DNA sequence: a glycine-to-glutamate substitution at position 40 and an aspartate-to-asparagine substitution at position 50. Our results further showed that lysine 13 and lysine 115 were methylated normally in cam12. It is likely that the electrophysiological abnormalities of cam12 are a direct reflection of the amino-acid substitutions, as opposed to improper posttranslational modification.     

NMR study of the interaction between the lac repressor and the lac operator

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.     

23Na NMR studies of rat outer medullary kidney tubules

Two reservations have previously made interpretation of biological 23Na NMR measurements difficult: the "size" of the extracellular space penetrated by the shift reagent and the possibility of a 60% reduction in the intensity of the NMR-visible 23Na signal due to quadrupolar interactions (Berendsen, H. J. C., and Edzes, H. T. (1973) Ann. N. Y. Acad. Sci. 204, 459-485; Civan, M. M., Degani, H., Margalit, Y., and Shporer, M. (1983) Am. J. Physiol. 245, C213-C219; Gupta, R. K., and Gupta, P. (1982) J. Magn. Reson. 47, 344-350). We have addressed both these issues using a suspension of rat outer medullary kidney tubules, nephron segments responsible for the fine control of total body volume and electrolyte balance. First, the extracellular space penetrated by the shift reagent dysprosium tripolyphosphate, as defined by the extracellular 23Na resonance, revealed a space similar to that which contained extracellular 35Cl- ions. Measurement of an extracellular 35Cl- space using 35Cl NMR was possible because the intracellular 35Cl- resonance was broadened beyond detection in the cells studied. Second, to characterize the reduction of the 23Na signal by quadrupolar interactions, the intracellular 23Na level was raised artificially by simultaneously inhibiting Na+ efflux and increasing the ion permeability of the plasma membrane. Under these conditions, NMR-observable intracellular Na+ reached a level which was approximately 81% of that in the medium, a level determined using chemical techniques. This observation would suggest that the resonance of the intracellular 23Na pool was not subject to a 60% reduction in signal intensity, as a result of nuclear quadrupolar interaction. The intracellular 23Na level measured, under basal conditions, was 23 +/- 2 mumol/ml of cell water (37 degrees C) (n = 3, S.D.) and was demonstrated to be responsive to a number of physiological stimuli. The level was temperature-sensitive. It was reduced by inhibitors of apical Na+ transport, furosemide and amiloride, and it was raised with (Na+ + K+)-ATPase inhibition. The furosemide and amiloride actions described would suggest that the Na+-transporting mechanisms sensitive to these agents (e.g. Na+/K+/Cl- cotransport system, Na+:H+ exchange system) contribute to the regulation of the intracellular Na+ level in the kidney tubular preparation studied.     

23Na NMR evaluation of human erythrocytes Na+/K+/CL-cotransport. A study in elderly hypertensives.

The behaviour of sodium transport systems across the cell membrane has been poorly investigated in elderly hypertension. Sodium efflux driven by Na+/K+/Cl-cotransport activity was therefore investigated (using a novel NMR-spectroscopy method) in 5 elderly hypertensive males (mean age 78 +/- 5 years) and 5 normotensive controls (mean age 79 +/- 3 years). In order to exclude any change in cotransport activity secondary to metabolic abnormalities, both patients and controls were non-obese and had normal glucose and lipid metabolisms. The Na+/K+/Cl-cotransport evaluation was performed after three months of pharmacological wash-out, under a diet containing 120 mEq of Na+/day. The resulting data showed that Na+ efflux due to outward Na+/K+/Cl-cotransport was higher in hypertensive group than in the normotensive one (0.50 +/- 0.10 mmol Na+/l cells/hr. vs 0.33 +/- 0.03 mmol Na+/l cells/hr., respectively, p < 0.05). Intracellular Na+ content was similar in both groups. At variance with previous data from the literature, our findings indicate that the Na+/K+/Cl-cotransport activity is elevated in elderly hypertensives.     

Solution NMR study of the interaction between NTF2 and nucleoporin FxFG repeats

Interactions with nucleoporins containing FxFG repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2). We describe here a solution NMR-based study that identifies primary and secondary FxFG-binding sites on NTF2 and accounts for a range of observations on the rate of NTF2 nuclear trafficking. We used three complementary NMR methods, namely amide group chemical shift titrations, NOE and cross-saturation measurements, to show that the major FxFG-binding site on the dimeric rat NTF2 (rNTF2) molecule is centred on Trp7 and is formed by residues from both NTF2 chains. A secondary FxFG-binding site is located at the rNTF2 hydrophobic cavity and these two sites, together with a surface hydrophobic cluster centred on Trp112, merge into an elongated hydrophobic stripe on the rNTF2 surface. The primary site centred on Trp7 is lost in the rNTF2-W7A mutant that has been shown to bind FxFG nucleoporins with greatly reduced affinity, whereas the secondary site at the rNTF2 hydrophobic cavity is retained. The interface between NTF2 and FxFG nucleoporins detected by NMR is more extensive than that detected by X-ray crystallography, and the presence of a secondary site at the NTF2 hydrophobic cavity accounts for the unexpectedly rapid nuclear import of rNTF2-W7R recently observed by others. The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking.     

NMR characterization of the interaction between the PUB domain of peptide:N-glycanase and ubiquitin-like domain of HR23

PUB domains are identified in several proteins functioning in the ubiquitin (Ub)-proteasome system and considered as p97-binding modules. To address the further functional roles of these domains, we herein characterized the interactions of the PUB domain of peptide:N-glycanase (PNGase) with Ub and Ub-like domain (UBL) of the proteasome shuttle factor HR23. NMR data indicated that PNGase-PUB exerts an acceptor preferentially for HR23-UBL, electrostatically interacting with the UBL surface employed for binding to other Ub/UBL motifs. Our findings imply that PNGase-PUB serves not only as p97-binding module but also as a possible activator of HR23 in endoplasmic reticulum-associated degradation mechanisms.