Nanosecond Dynamics at Protein Metal Sites: An Application of Perturbed Angular Correlation (PAC) of γ-Rays Spectroscopy.

Metalloproteins are essential to numerous reactions in nature, and constitute approximately one-third of all known proteins. Molecular dynamics of proteins has been elucidated with great success both by experimental and theoretical methods, revealing atomic level details of function involving the organic constituents on a broad spectrum of time scales. However, the characterization of dynamics at biomolecular metal sites on nanosecond time scales is scarce in the literature. The aqua ions of many biologically relevant metal ions exhibit exchange of water molecules on the nanosecond time scale or faster, often defining their reactivity in aqueous solution, and this is presumably also a relevant time scale for the making and breaking of coordination bonds between metal ions and ligands at protein metal sites. Ligand exchange dynamics is critical for a variety of elementary steps of reactions in metallobiochemistry, for example, association and dissociation of metal bound water, association of substrate and dissociation of product in the catalytic cycle of metalloenzymes, at regulatory metal sites which require binding and dissociation of metal ions, as well as in the transport of metal ions across cell membranes or between proteins involved in metal ion homeostasis. In Perturbed Angular Correlation of γ-rays (PAC) spectroscopy, the correlation in time and space of two γ-rays emitted successively in a nuclear decay is recorded, reflecting the hyperfine interactions of the PAC probe nucleus with the surroundings. This allows for characterization of molecular and electronic structure as well as nanosecond dynamics at the PAC probe binding site. Herein, selected examples describing the application of PAC spectroscopy in probing the dynamics at protein metal sites are presented, including (1) exchange of Cd2+ bound water in de novo designed synthetic proteins, and the effect of remote mutations on metal site dynamics; (2) dynamics at the ß-lactamase active site, where the metal ion appears to jump between the two adjacent sites; (3) structural relaxation in small blue copper proteins upon 111Ag+ to 111Cd2+ transformation in radioactive nuclear decay; (4) metal ion transfer between two HAH1 proteins with change in coordination number; and (5) metal ion sensor proteins with two coexisting metal site structures. With this Account, we hope to make our modest contribution to the field and perhaps spur additional interest in dynamics at protein metal sites, which we consider to be severely underexplored. Relatively little is known about detailed atomic motions at metal sites, for example, how ligand exchange processes affect protein function, and how the amino acid composition of the protein may control this facet of metal site characteristics. We also aim to provide the reader with a qualitative impression of the possibilities offered by PAC spectroscopy in bioinorganic chemistry, especially when elucidating dynamics at protein metal sites, and finally present data that may serve as benchmarks on a relevant time scale for development and tests of theoretical molecular dynamics methods applied to biomolecular metal sites.

Amyloid-ß and α-Synuclein Decrease the Level of Metal-Catalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing.

The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-ß (Aß) and α-synuclein (αS), associated with Alzheimer's and Parkinson's diseases, respectively, on the Cu(2+)-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu(2+) is bound to Aß or αS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aß or αS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu(2+) and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures.

Aggregation-Prone Amyloid-ß⋠Cu(II) Species Formed on the Millisecond Timescale under Mildly Acidic Conditions.

Metal ions and their interaction with the amyloid beta (Aß) peptide might be key elements in the development of Alzheimer's disease. In this work the effect of Cu(II) on the aggregation of Aß is explored on a timescale from milliseconds to days, both at physiological pH and under mildly acidic conditions, by using stopped-flow kinetic measurements (fluorescence and light-scattering), (1) H NMR relaxation and ThT fluorescence. A minimal reaction model that relates the initial Cu(II) binding and Aß folding with downstream aggregation is presented. We demonstrate that a highly aggregation prone Aß⋅Cu(II) species is formed on the sub-second timescale at mildly acidic pH. This observation might be central to the molecular origin of the known detrimental effect of acidosis in Alzheimer's disease.

The pKa value and accessibility of cysteine residues are key determinants for protein substrate discrimination by glutaredoxin.

The enzyme glutaredoxin catalyzes glutathione exchange, but little is known about its interaction with protein substrates. Very different proteins are substrates in vitro, and the enzyme seems to have low requirements for specific protein interactions. Here we present a systematic investigation of the interaction between human glutaredoxin 1 and glutathionylated variants of a single model protein. Thus, single cysteine variants of acyl-coenzyme A binding protein were produced creating a set of substrates in the same protein background. The rate constants for deglutathionylation differ by more than 2 orders of magnitude between the best (k1 = 1.75 × 10(5) M(-1) s(-1)) and the worst substrate (k1 = 4 × 10(2) M(-1) s(-1)). The pKa values of the substrate cysteine residues were determined by NMR spectroscopy and found to vary from 8.2 to 9.9. Rates of glutaredoxin 1-catalyzed deglutathionylation were assessed with respect to substrate cysteine pKa values, cysteine residue accessibility, local stability, and backbone dynamics. Good substrates are characterized by a combination of high accessibility of the glutathionylated site and low pKa of the cysteine residue.

Analysis of protein aggregation in neurodegenerative disease.

Pathological protein and peptide aggregation are key events in a number of chronic and devastating neurodegenerative conditions including dementias such as Alzheimer's and Creutzfeldt-Jakob's disease and other central nervous system diseases such as Parkinson's and Huntington's disease and amyotrophic lateral sclerosis. Analytical methods for studying protein aggregation in these diseases are important for mapping pathophysiological events and ultimately for the development of new therapies and better diagnostic tools.

Rapid exchange of metal between Zn(7)-metallothionein-3 and amyloid-ß peptide promotes amyloid-related structural changes.

Metal ions, especially Zn(2+) and Cu(2+), are implemented in the neuropathogenesis of Alzheimer's disease (AD) by modulating the aggregation of amyloid-ß peptides (Aß). Also, Cu(2+) may promote AD neurotoxicity through production of reactive oxygen species (ROS). Impaired metal ion homeostasis is most likely the underlying cause of aberrant metal-Aß interaction. Thus, focusing on the body's natural protective mechanisms is an attractive therapeutic strategy for AD. The metalloprotein metallothionein-3 (MT-3) prevents Cu-Aß-mediated cytotoxicity by a Zn-Cu exchange that terminates ROS production. Key questions about the metal exchange mechanisms remain unanswered, e.g., whether an Aß-metal-MT-3 complex is formed. We studied the exchange of metal between Aß and Zn(7)-MT-3 by a combination of spectroscopy (absorption, fluorescence, thioflavin T assay, and nuclear magnetic resonance) and transmission electron microscopy. We found that the metal exchange occurs via free Cu(2+) and that an Aß-metal-MT-3 complex is not formed. This means that the metal exchange does not require specific recognition between Aß and Zn(7)-MT-3. Also, we found that the metal exchange caused amyloid-related structural and morphological changes in the resulting Zn-Aß aggregates. A detailed model of the metal exchange mechanism is presented. This model could potentially be important in developing therapeutics with metal-protein attenuating properties in AD.

Cu(II) mediates kinetically distinct, non-amyloidogenic aggregation of amyloid-beta peptides.

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-ß (Aß) peptide. Elucidating the underlying Cu(II)-induced Aß aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aß(1-40) using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aß ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aß, because three different kinetic scenarios were observed depending on the Cu(II):Aß ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aß ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aß ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aß(1-42) were compatible with the conclusions for Aß(1-40) even though the low solubility of Aß(1-42) precluded examination under the same conditions as for the Aß(1-40). Experiments with Aß(1-16) and Aß(1-28) showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aß aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aß(1-40) depending on the Cu(II):Aß ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.

A new water-soluble Cu(II) chelator that retrieves Cu from Cu(amyloid-ß) species, stops associated ROS production and prevents Cu(II)-induced Aß aggregation.

The synthesis of the H(2)L(2-) ligand (N,N'-Bis[(5-sulfonato-2-hydroxy)benzyl]-N,N'-dimethyl-ethane-1,2-diamine) and characterizations of the corresponding Cu(II) complex [Cu(L)(H(2)O)](2-) (1) by X-ray diffraction, EPR, UV-Visible and potentiometry are described. At pH 7.4, the affinity of Cu(II) for this ligand is approximately 4 × 10(14)M(-1). Coordination of redox active metal ions such as copper or iron to the amyloid-ß (Aß) peptide has been linked to deleterious processes encountered in the etiology of Alzheimer disease (AD), such as Aß aggregation and reactive oxygen species (ROS) production. In this context, the ability of the H(2)L(2-) to extract Cu(II) from Cu(Aß) species where Aß is the peptide involved in AD, is reported as well as its capacity to redox silence the Cu(Aß) induced ROS formation and to prevent Cu(II)-induced Aß aggregation. Such water soluble sulfonato-derivatives of Cu(II) chelators are very interesting counterparts for in vitro study of chelators' properties required to attend further biological applications as therapeutic tools against AD.