Magnesium(II)-ATP Complexes in 1-Ethyl-3-Methylimidazolium Acetate Solutions Characterized by 31 Mg ß-Radiation-Detected NMR Spectroscopy.

The complexation of MgII with adenosine 5'-triphosphate (ATP) is omnipresent in biochemical energy conversion, but is difficult to interrogate directly. Here we use the spin- 1/2 ß-emitter 31 Mg to study MgII -ATP complexation in 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) solutions using ß-radiation-detected nuclear magnetic resonance (ß-NMR). We demonstrate that (nuclear) spin-polarized 31 Mg, following ion-implantation from an accelerator beamline into EMIM-Ac, binds to ATP within its radioactive lifetime before depolarizing. The evolution of the spectra with solute concentration indicates that the implanted 31 Mg initially bind to the solvent acetate anions, whereafter they undergo dynamic exchange and form either a mono- (31 Mg-ATP) or di-nuclear (31 MgMg-ATP) complex. The chemical shift of 31 Mg-ATP is observed up-field of 31 MgMg-ATP, in accord with quantum chemical calculations. These observations constitute a crucial advance towards using ß-NMR to probe chemistry and biochemistry in solution.

Probing the Secondary Structure of Individual Aß40 Amorphous Aggregates and Fibrils by AFM-IR Spectroscopy.

Structural characterization of aggregates and fibrils of the Aß protein is pivotal to the molecular-level elucidation of Alzheimer's disease (AD). AFM-IR spectroscopy provides nanoscale resolution, and thus allows the interrogation of individual aggregates and fibrils. During aggregation of Aß, we observed mainly disordered Aß at t=15 min, but substantial structural diversity including the co-existence of parallel and antiparallel ß-sheets within a large amorphous aggregate at t=2 hours, while fibrils exhibited the expected signature of parallel ß-sheets at t=1 week. The resonance observed for parallel ß-sheets at t=2 hours coincides with that observed for fibrils (at 1634 cm-1 ), thus indicating that fibril-like species exist within the large aggregates. Therefore, nucleation might occur within such species, in analogy to current theories of protein crystallization in which nucleation occurs within large protein clusters. Cu2+ perturbs Aß aggregation, catalysing rapid formation of amorphous aggregates with diverse secondary structure, but inhibiting fibril growth.

Direct observation of Mg2+ complexes in ionic liquid solutions by 31Mg ß-NMR spectroscopy.

NMR spectra of Mg2+ ions in ionic liquids were recorded using a highly sensitive variant of NMR spectroscopy known as ß-NMR. The ß-NMR spectra of MgCl2 in EMIM-Ac and EMIM-DCA compare favourably with conventional NMR, and exhibit linewidths of ∼3 ppm, allowing for discrimination of species with oxygen and nitrogen coordination.

The Pathogenic A2V Mutant Exhibits Distinct Aggregation Kinetics, Metal Site Structure, and Metal Exchange of the Cu2+ -Aß Complex.

A prominent current hypothesis is that impaired metal ion homeostasis may contribute to Alzheimer's disease (AD). We elucidate the interaction of Cu2+ with wild-type (WT) Aß1-40 and the genetic variants A2T and A2V which display increasing pathogenicity as A2T

Specificity of the Metalloregulator CueR for Monovalent Metal Ions: Possible Functional Role of a Coordinated Thiol?

Metal-ion-responsive transcriptional regulators within the MerR family effectively discriminate between mono- and divalent metal ions. Herein we address the origin of the specificity of the CueR protein for monovalent metal ions. Several spectroscopic techniques were employed to study Ag(I) , Zn(II) , and Hg(II) binding to model systems encompassing the metal-ion-binding loop of CueR from E. coli and V. cholerae. In the presence of Ag(I) , a conserved cysteine residue displays a pKa  value for deprotonation of the thiol that is close to the physiological pH value. This property is only observed with the monovalent metal ion. Quantum chemically optimized structures of the CueR metal site with Cys 112 protonated demonstrate that the conserved Ser 77 backbone carbonyl oxygen atom from the other monomer of the homodimer is "pulled" towards the metal site. A common allosteric mechanism of the metalloregulatory members of the MerR family is proposed. For CueR, the mechanism relies on the protonation of Cys 112.

Zn(II) and Hg(II) binding to a designed peptide that accommodates different coordination geometries.

Designed metal ion binding peptides offer a variety of applications in both basic science as model systems of more complex metalloproteins, and in biotechnology, e.g. in bioremediation of toxic metal ions, biomining or as artificial enzymes. In this work a peptide (HS: Ac-SCHGDQGSDCSI-NH2) has been specifically designed for binding of both Zn(II) and Hg(II), i.e. metal ions with different preferences in terms of coordination number, coordination geometry, and to some extent ligand composition. It is demonstrated that HS accommodates both metal ions, and the first coordination sphere, metal ion exchange between peptides, and speciation are characterized as a function of pH using UV-absorption-, synchrotron radiation CD-, (1)H-NMR-, and PAC-spectroscopy as well as potentiometry. Hg(II) binds to the peptide with very high affinity in a {HgS2} coordination geometry, bringing together the two cysteinates close to each end of the peptide in a loop structure. Despite the high affinity, Hg(II) is kinetically labile, exchanging between peptides on the subsecond timescale, as indicated by line broadening in (1)H-NMR. The Zn(II)-HS system displays more complex speciation, involving monomeric species with coordinating cysteinates, histidine, and a solvent water molecule, as well as HS-Zn(II)-HS complexes. In summary, the HS peptide displays conformational flexibility, contains many typical metal ion binding groups, and is able to accommodate metal ions with different structural and ligand preferences with high affinity. As such, the HS peptide may be a scaffold offering binding of a variety of metal ions, and potentially serve for metal ion sequestration in biotechnological applications.

Billion-fold enhancement in sensitivity of nuclear magnetic resonance spectroscopy for magnesium ions in solution.

ß-nuclear magnetic resonance (NMR) spectroscopy is highly sensitive compared to conventional NMR spectroscopy, and may be applied for several elements across the periodic table. ß-NMR has previously been successfully applied in the fields of nuclear and solid-state physics. In this work, ß-NMR is applied, for the first time, to record an NMR spectrum for a species in solution. (31)Mg ß-NMR spectra are measured for as few as 10(7) magnesium ions in ionic liquid (EMIM-Ac) within minutes, as a prototypical test case. Resonances are observed at 3882.9 and 3887.2 kHz in an external field of 0.3 T. The key achievement of the current work is to demonstrate that ß-NMR is applicable for the analysis of species in solution, and thus represents a novel spectroscopic technique for use in general chemistry and potentially in biochemistry.

On the possible roles of N-terminal His-rich domains of Cu,Zn SODs of some Gram-negative bacteria.

The Cu,Zn superoxide dismutases (Cu,Zn SOD) isolated from some Gram-negative bacteria possess a His-rich N-terminal metal binding extension. The N-terminal domain of Haemophilus ducreyi Cu,Zn SOD has been previously proposed to play a copper(II)-, and may be a zinc(II)-chaperoning role under metal ion starvation, and to behave as a temporary (low activity) superoxide dismutating center if copper(II) is available. The N-terminal extension of Cu,Zn SOD from Actinobacillus pleuropneumoniae starts with an analogous sequence (HxDHxH), but contains considerably fewer metal binding sites. In order to study the possibility of the generalization of the above mentioned functions over all Gram-negative bacteria possessing His-rich N-terminal extension, here we report thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first eight amino acids (HADHDHKK-NH(2), L) of the enzyme isolated from A. pleuropneumoniae. In equimolar solutions of Cu(II)/Zn(II) and the peptide the MH(2)L complexes are dominant in the neutral pH-range. L has extraordinary copper(II) sequestering capacity (K(D,Cu)=7.4×10(-13)M at pH 7.4), which is provided only by non-amide (side chain) donors. The central ion in CuH(2)L is coordinated by four nitrogens {NH(2),3N(im)} in the equatorial plane. In ZnH(2)L the peptide binds to zinc(II) through a {NH(2),2N(im),COO(-)} donor set, and its zinc binding affinity is relatively modest (K(D,Zn)=4.8×10(-7)M at pH 7.4). Consequently, the presented data do support a general chaperoning role of the N-terminal His-rich region of Gram-negative bacteria in copper(II) uptake, but do not confirm similar function for zinc(II). Interestingly, the complex CuH(2)L has very high SOD-like activity, which may further support the multifunctional role of the copper(II)-bound N-terminal His-rich domain of Cu,Zn SODs of Gram-negative bacteria. The proposed structure for the MH(2)L complexes has been verified by semiempirical quantum chemical calculations (PM6), too.

Towards the role of metal ions in the structural variability of proteins: CdII speciation of a metal ion binding loop motif.

A de novo designed dodecapeptide (HS), inspired by the metal binding loops of metal-responsive transcriptional activators, was synthesized. The aim was to create a model system for structurally promiscuous and intrinsically unstructured proteins, and explore the effect of metal ions on their structure and dynamics. The interaction with Cd(II) was investigated by UV, synchrotron radiation CD, (1)H NMR, and perturbed angular correlation (PAC) of γ-rays spectroscopy, pH-potentiometry, and molecular modelling. The peptide mainly displays characteristics of random coil in the CD spectra, and the molecular dynamics simulations demonstrate that it is unstructured with transient and varying helical content. The spectroscopic studies revealed the formation of loop structures with the coordination of the two Cys-thiolates close to each end of the HS peptide, in the presence of one equivalent of Cd(II) per ligand. The imidazole moiety from histidine is also bound to Cd(II) at neutral pH and above. In the presence of 0.5 equivalent of Cd(II) per HS metal bridged structures with e.g. CdS(2)N(2) and possibly CdS(4) coordination geometries are formed above pH ~6. In an equilibrium of several co-existing species the peptide is exchanging between a number of structures also in its metal ion bound state(s), as indicated by NMR and PAC data.