Search

L-RNA aptamers were developed that bind to barnase RNase and thereby inhibit the function of the enzyme. These aptamers were obtained by first carrying out in vitro selection of D-RNAs that bind to the full-length synthetic D-enantiomer of barnase, then reversing the mirror and preparing L-RNAs of identical sequence that similarly bind to natural L-barnase. The resulting L-aptamers bind L-barnase with an affinity of ∼100 nM and function as competitive inhibitors of enzyme cleavage of D-RNA substrates. L-RNA aptamers are resistant to degradation by ribonucleases, thus enabling them to function in biological samples, most notably for applications in molecular diagnostics and therapeutics. In addition to the irony of using RNA to inhibit RNase, L-RNA aptamers such as those described here could be used to measure the concentration or inhibit the function of RNase in the laboratory or in biological systems., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A general analytical method for the detection of target ligands has been developed, based on a special class of self-replicating aptazymes. These "autocatalytic aptazymes" are generated by linking an aptamer domain to the catalytic domain of a self-replicating RNA enzyme. Ligand-dependent self-replication of RNA proceeds in a self-sustained manner, undergoing exponential amplification at a constant temperature without the assistance of any proteins or other biological materials. The rate of exponential amplification is dependent on the concentration of the ligand, thus enabling quantitative ligand detection. This system has the potential to detect any ligand that can be recognized by an aptamer, including small molecules and proteins. The instability of RNA in biological samples due to the presence of ribonucleases can be overcome by employing the enantiomeric L-RNA form of the self-replicating enzyme. Methods for real-time fluorescence monitoring over the course of exponential amplification are currently being developed., (© 2015 Elsevier Inc. All rights reserved.)

A nuclease-resistant RNA enzyme, constructed entirely from L-ribonucleotides, was shown to undergo ligand-dependent, self-sustained replication with exponential growth. The catalytic motif is based on a previously described RNA ligase that can undergo either self- or cross-replication but had been limited in its application to ligand sensing due to its susceptibility to degradation by ribonucleases. The self-replicating RNA enzyme and its RNA substrates were prepared synthetically from either D- or L-nucleoside phosphoramidites. The D and L reaction systems undergo isothermal, ligand-dependent exponential amplification in the same manner, but only the l system is impervious to ribonucleases and can operate, for example, in the presence of human serum. This system has potential for the quantitative detection of various ligands that are present within biological or environmental samples. In addition, this work provides the first demonstration of the self-sustained exponential amplification of nonbiological molecules.

Hemoproteins are ubiquitous in biology and are commonly involved in critical processes such as electron transfer, oxidative phosphorylation, and signal transduction. Both the protein environment and the heme cofactor contribute to generate the range of chemical properties needed for these diverse functions. Using the heme nitric oxide/oxygen binding (H-NOX) protein from the thermophilic bacterium Thermoanaerobacter tengcongensis, we have shown that heme electronic properties can be modulated by porphyrin distortion within the same protein scaffold without changing the heme ligation state or heme environment. The degree of heme distortion was found to be directly correlated to the electron density at the heme iron, as evidenced by dramatic changes in the heme redox potential and pK(a) of the distal ligand ((-)OH vs H(2)O). Protein-induced porphyrin distortion represents a new strategy to rationally tune the electronic properties of protein-bound porphyrins and could be used to engineer proteins with desired reactivity or functionality.

Globins are heme-containing proteins that are best known for their roles in oxygen (O(2)) transport and storage. However, more diverse roles of globins in biology are being revealed, including gas and redox sensing. In the nematode Caenorhabditis elegans, 33 globin or globin-like genes were recently identified, some of which are known to be expressed in the sensory neurons of the worm and linked to O(2) sensing behavior. Here, we describe GLB-6, a novel globin-like protein expressed in the neurons of C. elegans. Recombinantly expressed full-length GLB-6 contains a heme site with spectral features that are similar to those of other bis-histidyl ligated globins, such as neuroglobin and cytoglobin. In contrast to these globins, however, ligands such as CO, NO, and CN(-) do not bind to the heme in GLB-6, demonstrating that the endogenous histidine ligands are likely very tightly coordinated. Additionally, GLB-6 exhibits rapid two-state autoxidation kinetics in the presence of physiological O(2) levels as well as a low redox potential (-193 +/- 2 mV). A high-resolution (1.40 A) crystal structure of the ferric form of the heme domain of GLB-6 confirms both the putative globin fold and bis-histidyl ligation and also demonstrates key structural features that can be correlated with the unusual ligand binding and redox properties exhibited by the full-length protein. Taken together, the biochemical properties of GLB-6 suggest that this neural protein would most likely serve as a physiological sensor for O(2) in C. elegans via redox signaling and/or electron transfer.

Hemoprotein-based scaffolds containing phosphorescent ruthenium(II) CO mesoporphyrin IX (RuMP) are reported here for oxygen (O(2)) sensing in biological contexts. RuMP was incorporated into the protein scaffolds during protein expression utilizing a novel method that we have described previously. A high-resolution (2.00 A) crystal structure revealed that the unnatural porphyrin binds to the proteins in a manner similar to the native heme and does not perturb the protein fold. The protein scaffolds were found to provide unique coordination environments for RuMP and modulate the porphyrin emission properties. Emission lifetime measurements demonstrate a linear O(2) response within the physiological range and precision comparable to commercial O(2) sensors. The RuMP proteins are robust, readily modifiable platforms and display promising O(2) sensing properties for future in vivo applications.

Nitric oxide (NO) signaling in mammals controls important processes such as smooth muscle relaxation and neurotransmission by the activation of soluble guanylate cyclase (sGC). NO binding to the heme domain of sGC leads to dissociation of the iron-histidine (Fe-His) bond, which is required for enzyme activity. The heme domain of sGC belongs to a larger class of proteins called H-NOX (Heme-Nitric oxide/OXygen) binding domains. Previous crystallographic studies on H-NOX domains demonstrate a correlation between heme bending and protein conformation. It was unclear, however, whether these structural changes were important for signal transduction. Subsequent NMR solution structures of H-NOX proteins show a conformational change upon disconnection of the heme and proximal helix, similar to those observed in the crystallographic studies. The atomic details of these conformational changes, however, are lacking in the NMR structures especially at the heme pocket. Here, a high-resolution crystal structure of an H-NOX mutant mimicking a broken Fe-His bond is reported. This mutant exhibits specific changes in heme conformation and major N-terminal displacements relative to the wild-type H-NOX protein. Fe-His ligation is ubiquitous in all H-NOX domains, and therefore, the heme and protein conformational changes observed in this study are likely to occur throughout the H-NOX family when NO binding leads to rupture of the Fe-His bond.

Phenoxazinone synthase, an oligomeric multicopper oxidase produced by Streptomyces antibioticus, is responsible for the six-electron oxidative coupling of two molecules of 4-methyl 3-hydroxyanthraniloyl pentapeptide to form the phenoxazinone chromophore of the antineoplastic agent actinomycin D. Spectroscopic studies have shown that the enzyme contains one type I (blue) and three to four type II copper centers. However, the exact arrangement of the copper centers in this multicopper oxidase is unknown. As a first step towards determining the three-dimensional structure of the enzyme, phenoxazinone synthase has been crystallized. The hexameric form of phenoxazinone synthase was purified from 72 h cultures of S. lividans containing the plasmid pIJ702. Purified hexamers were concentrated to 75 mg ml(-1) and used to grow two forms of crystals. Data collected from the two crystal forms were processed in two separate space groups. Crystals of both forms were grown at 288 K using the sitting-drop vapour-diffusion method. Native data sets extending to resolutions of 3.35 and 2.30 A have been collected and processed in space groups R32 and P1, respectively.