We have used two-dimensional 'H NMR spectroscopy to study the conformation of the thrombin-binding aptamer d (GGTTGGTGTGGTTGG) in solution. This is one of a series of thrombin-binding DNA aptamers with a consensus 15-base sequence that was recently isolated and shown to inhibit thrombin-catalyzed fibrin clot formation in vitro [Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. (1992) Nature (London) 355,[564][565][566]. The oligonucleotide forms a unimolecular DNA quadruplex consisting of two G-quartets connected by two TT loops and one TGT loop. A potential T-T bp is formed between the two TT loops across the diagonal of the top G-quartet. Thus, all of the invariant bases in the consensus sequence are base-paired. This aptamer structure was determined by NMR and illustrates that this molecule forms a specific folded structure. Knowledge of this structure may be used in the further development of oligonucleotide-based thrombin inhibitors.The ability of nucleic acids to fold into a variety of different structures has been exploited in the development of techniques for the isolation of aptamers (1) which are DNA or RNA oligonucleotides that have been screened from a randomly generated population of sequences for their ability to bind a desired molecular target (2-4). The isolation process involves repeated cycles of selection for, and enrichment of, oligonucleotides with an affinity to a specific target, followed by amplification of these sequences using the PCR (5). Oligonucleotides with the selected characteristics-i.e., binding to a specific molecule-are finally identified through cloning and sequencing. The potential of these methods for the development of oligonucleotide-based therapeutics has recently been explored by their application in isolating oligonucleotide ligands that selectively inhibit the activity of a target protein. Selection of DNA aptamers that bind to thrombin and inhibit thrombin-catalyzed fibrin-clot formation in vitro (6) and more recently an RNA oligonucleotide that specifically inhibits cDNA synthesis by human immunodeficiency virus reverse transcriptase in vitro (7) have been reported.Here we report two-dimensional NMR studies of the thrombin-binding aptamer d(GGTTGGTGTGGTTGG) (thrombin aptamer) that conforms to the consensus sequence d(GGtTGGN2_5GGtTGG), where an uppercase letter indicates an invariant base, a lowercase letter indicates a base bias at that position, and there are usually three central N nucleotides (6). The oligonucleotide folds into a unimolecular quadruplex containing two G-quartets (8) linked by two TT loops at one end and a TGT loop at the other end. The invariant thymines in the TT loops are potentially basepaired across the top of one G-quartet. This aptamer structure was determined by NMR techniques and illustrates that this molecule forms a specific folded structure. Knowledge of this structure should be useful in the further development of oligonucleotide-based therapeutics or as a starting point for small-molecule drug design...

A subset of individuals with familial amyotrophic lateral sclerosis (FALS) possesses dominantly inherited mutations in the gene that encodes copper-zinc superoxide dismutase (CuZnSOD). A4V and G93A, two of the mutant enzymes associated with FALS, were shown to catalyze the oxidation of a model substrate (spin trap 5,5'-dimethyl-1-pyrroline N-oxide) by hydrogen peroxide at a higher rate than that seen with the wild-type enzyme. Catalysis of this reaction by A4V and G93A was more sensitive to inhibition by the copper chelators diethyldithiocarbamate and penicillamine than was catalysis by wild-type CuZnSOD. The same two chelators reversed the apoptosis-inducing effect of mutant enzymes expressed in a neural cell line. These results suggest that oxidative reactions catalyzed by mutant CuZnSOD enzymes initiate the neuropathologic changes in FALS.

We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of "WT-like" FALS mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133⌬) conferred a similar degree of incremental stabilization as did metal ion binding to WT SOD1. However, these mutants were all destabilized by ϳ1-6°C compared with the corresponding WT SOD1 species. Most of the "metal binding region" FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions that probably resulted from unfolding of metal-free species at ϳ4 -12°C below the observed melting of the least stable WT species. We conclude that decreased conformational stability shared by all of these mutant SOD1s may contribute to SOD1 toxicity in FALS.Copper/zinc superoxide dismutase (SOD1) 1 catalyzes the disproportionation of two molecules of superoxide anion (O 2 . )into O 2 and H 2 O 2 (1, 2) in all eukaryotic cells. Many specific, highly conserved structural interactions confer upon SOD1 a remarkable thermal stability (3-6) and resistance to chemical denaturation (7-9).Each subunit of homodimeric SOD1 is built upon a flattened ␤-barrel motif with additional loop regions that contribute to metal ion binding and formation of the active site (10). One catalytic copper ion and one buried zinc ion per subunit are bound at the active site on the external surface of the ␤-barrel. Occupancy of the metal ion binding sites confers greater thermal stabilization to the bovine SOD1 apoenzyme (3, 4). The copper and zinc ions are linked directly via the imidazolate side chain of the shared His-63 residue 2 and indirectly via extended interactions between their respective ligands. SOD1 dimerization is stabilized by optimized hydrophobic interactions at the contact interface between complementary patches on each subunit (10 -12). A conserved intrasubunit disulfide bond involving Cys-57 also stabilizes the enzyme by anchoring a loop that forms part of the dimer interface to the ␤-barrel at Cys-146.A subset of SOD1 mutations in familial amyotrophic lateral sclerosis (FALS) have been proposed to destabilize the ␤-barrel or disrupt dimerization of SOD1 monomers (13,14). A crystal structure obtained for the G37R SOD1 mutant shows minimal perturbation of the averaged backbone conformation but exhibits unusually high atomic displacement parameters, suggestive of increased molecular flexibility in some regions of the molecule (15). Consistent with this, some mutant SOD1s exhibit acceler...

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A series of mutant human and yeast copperzinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.