Nathan P. Cook

Coal as an abundant source of graphene quantum dots

Coal is the most abundant and readily combustible energy resource being used worldwide. However, its structural characteristic creates a perception that coal is only useful for producing energy via burning. Here we report a facile approach to synthesize tunable graphene quantum dots from various types of coal, and establish that the unique coal structure has an advantage over pure sp2-carbon allotropes for producing quantum dots. The crystalline carbon within the coal structure is easier to oxidatively displace than when pure sp2-carbon structures are used, resulting in nanometre-sized graphene quantum dots with amorphous carbon addends on the edges. The synthesized graphene quantum dots, produced in up to 20% isolated yield from coal, are soluble and fluorescent in aqueous solution, providing promise for applications in areas such as bioimaging, biomedicine, photovoltaics and optoelectronics, in addition to being inexpensive additives for structural composites.

Sensing Amyloid-β Aggregation Using Luminescent Dipyridophenazine Ruthenium(II) Complexes

The aggregation of amyloid-β (Aβ) peptides has been associated with the onset of Alzheimers disease. Here, we report the use of a luminescent dipyridophenazine ruthenium(II) complex to monitor Aβ fibrillization. This complex is not photoluminescent in aqueous solution nor in the presence of monomeric Aβ, but it presents a strong photoluminescence in the presence of Aβ fibril aggregates. One of the advantages of this metal complex is its large Stokes shift (180 nm). Furthermore, the long-lived photoluminescence lifetime of this ruthenium complex allows its use for the detection of fibrillar proteins in the presence of short-lived fluorescent backgrounds, using time-gating technology. We will present evidence of the advantages of dipyridophenazine ruthenium(II) complexes for monitoring protein fibrillization in highly fluorescent media.

Unraveling the Photoluminescence Response of Light-Switching Ruthenium(II) Complexes Bound to Amyloid-β

Photoluminescent molecules are widely used for real-time monitoring of peptide aggregation. In this Article, we detail both experimental and computational modeling to elucidate the interaction between [Ru(bpy)2dppz](2+) and amyloid-β (Aβ(1-40)) aggregates. The transition from monomeric to fibrillar Aβ is of interest in the study of Alzheimers disease. Concentration-dependent experiments allowed the determination of a dissociation constant of 2.1 μM, while Job plots provided a binding stoichiometry of 2.6 Aβ monomers per [Ru(bpy)2dppz](2+). Our computational approach that combines molecular docking (both rigid and flexible) and all-atom molecular dynamics (MD) simulations predicts that the hydrophobic cleft between Val18 and Phe20 is a plausible binding site, which could also explain the increase in photoluminescence of [Ru(bpy)2dppz](2+) upon binding. This binding site is parallel to the fibril axis, in marked contrast to the binding site of these complexes in DNA (perpendicular to the DNA axis). Other binding sites may exist at the edges of the Aβ fibril, but they are actually of low abundance in an Aβ fibril several micrometers long. The assignment of the binding site was confirmed by binding studies in an Aβ fragment (Aβ(25-35)) that lacked the amino acids necessary to form the binding site. The agreement between the experimental and computational work is remarkable and provides a general model that can be used for studying the interaction of amyloid-binding molecules to Aβ.

Deamidation of asparagine to aspartate destabilizes Cu, Zn superoxide dismutase, accelerates fibrillization, and mirrors ALS-linked mutations.

The reactivity of asparagine residues in Cu, Zn superoxide dismutase (SOD1) to deamidate to aspartate remains uncharacterized; its occurrence in SOD1 has not been investigated, and the biophysical effects of deamidation on SOD1 are unknown. Deamidation is, nonetheless, chemically equivalent to Asn-to-Asp missense mutations in SOD1 that cause amyotrophic lateral sclerosis (ALS). This study utilized computational methods to identify three asparagine residues in wild-type (WT) SOD1 (i.e., N26, N131, and N139) that are predicted to undergo significant deamidation (i.e., to >20%) on time scales comparable to the long lifetime (>1 year) of SOD1 in large motor neurons. Site-directed mutagenesis was used to successively substitute these asparagines with aspartate (to mimic deamidation) according to their predicted deamidation rate, yielding: N26D, N26D/N131D, and N26D/N131D/N139D SOD1. Differential scanning calorimetry demonstrated that the thermostability of N26D/N131D/N139D SOD1 is lower than WT SOD1 by ~2-8 °C (depending upon the state of metalation) and <3 °C lower than the ALS mutant N139D SOD1. The triply deamidated analog also aggregated into amyloid fibrils faster than WT SOD1 by ~2-fold (p < 0.008**) and at a rate identical to ALS mutant N139D SOD1 (p > 0.2). A total of 534 separate amyloid assays were performed to generate statistically significant comparisons of aggregation rates among WT and N/D SOD1 proteins. Capillary electrophoresis and mass spectrometry demonstrated that ~23% of N26 is deamidated to aspartate (iso-aspartate was undetectable) in a preparation of WT human SOD1 (isolated from erythrocytes) that has been used for decades by researchers as an analytical standard. The deamidation of asparagine--an analytically elusive, sub-Dalton modification--represents a plausible and overlooked mechanism by which WT SOD1 is converted to a neurotoxic isoform that has a similar structure, instability, and aggregation propensity as ALS mutant N139D SOD1.

Arresting Amyloid with Coulomb’s Law: Acetylation of ALS-Linked SOD1 by Aspirin Impedes Aggregation

Although the magnitude of a proteins net charge (Z) can control its rate of self-assembly into amyloid, and its interactions with cellular membranes, the net charge of a protein is not viewed as a druggable parameter. This article demonstrates that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of superoxide dismutase (SOD1) by increasing the intrinsic net negative charge of the polypeptide, i.e., by acetylation (neutralization) of multiple lysines. The protective effects of acetylation were diminished (but not abolished) in 100 mM NaCl and were statistically significant: a total of 432 thioflavin-T amyloid assays were performed for all studied proteins. The acetylation of as few as three lysines by aspirin in A4V apo-SOD1-a variant that causes familial amyotrophic lateral sclerosis (ALS)-delayed amyloid nucleation by 38% and slowed amyloid propagation by twofold. Lysines in wild-type- and ALS-variant apo-SOD1 could also be peracetylated with aspirin after fibrillization, resulting in supercharged fibrils, with increases in formal net charge of ∼2 million units. Peracetylated SOD1 amyloid defibrillized at temperatures below unacetylated fibrils, and below the melting temperature of native Cu2,Zn2-SOD1 (e.g., fibril Tm = 84.49°C for acetylated D90A apo-SOD1 fibrils). Targeting the net charge of native or misfolded proteins with small molecules-analogous to how an enzymes Km or Vmax are medicinally targeted-holds promise as a strategy in the design of therapies for diseases linked to protein self-assembly.

Grb2 monomer–dimer equilibrium determines normal versus oncogenic function

The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

Ruthenium red colorimetric and birefringent staining of amyloid-β aggregates in vitro and in Tg2576 mice.

Alzheimers disease (AD) is a devastating neurodegenerative disease most notably characterized by the misfolding of amyloid-β (Aβ) into fibrils and its accumulation into plaques. In this Article, we utilize the affinity of Aβ fibrils to bind metal cations and subsequently imprint their chirality to bound molecules to develop novel imaging compounds for staining Aβ aggregates. Here, we investigate the cationic dye ruthenium red (ammoniated ruthenium oxychloride) that binds calcium-binding proteins, as a labeling agent for Aβ deposits. Ruthenium red stained amyloid plaques red under light microscopy, and exhibited birefringence under crossed polarizers when bound to Aβ plaques in brain tissue sections from the Tg2576 mouse model of AD. Staining of Aβ plaques was confirmed via staining of the same sections with the fluorescent amyloid binding dye Thioflavin S. In addition, it was confirmed that divalent cations such as calcium displace ruthenium red, consistent with a mechanism of binding by electrostatic interaction. We further characterized the interaction of ruthenium red with synthetic Aβ fibrils using independent biophysical techniques. Ruthenium red exhibited birefringence and induced circular dichroic bands at 540 nm upon binding to Aβ fibrils due to induced chirality. Thus, the chirality and cation binding properties of Aβ aggregates could be capitalized for the development of novel amyloid labeling methods, adding to the arsenal of AD imaging techniques and diagnostic tools.

Corrigendum: Coal as an abundant source of graphene quantum dots

Nature Communications 4: Article number: 2943 (2013); Published: 6 December 2013; Updated: 23 April 2015. In this Article, the bituminous coal graphene quantum dots (b-GQD) are described throughout this paper as having a crystalline hexagonal structure. Following further careful study of the high-resolution transmission electron microscopy data, this claim is too rigorous, and the b-GQDs instead should be referred to as crystalline.

Facile methodology for monitoring amyloid-β fibrillization.

Amyloid-β (Aβ) is a peptide fragment that is prone to aggregate into large fibrils under physiological conditions. Many techniques have been developed to quickly monitor the transition from a primarily monomeric peptide into fibrils. Here we propose a novel method for both incubating and monitoring changes in Aβ aggregation by using modified NMR tubes, a microtube thermoshaker, and a fluorescence or UV-vis spectrometer. These NMR tubes are thin and cylindrical, which allows efficient heat transfer and orbital shaking. Our results demonstrate that our technique is both reliable and expedient when tracking Aβ fibrillization using fluorescence or turbidity assays, which presents an alternative for laboratories without specialized equipment for incubating peptide.

Corrigendum: Grb2 monomer-dimer equilibrium determines normal versus oncogenic function.

Nature Communications 6, Article number: 7354 (2015); Published: 24 June 2015; Updated: 3 August 2015. In this Article, there are errors in the labelling of the y axes in Figs 2 and 3. In both figures, ‘Fluorescence intensity’ should read ‘Number of pixels’. The correct versions of these figures appear below.