NMR structure of an acyl-carrier protein from Borrelia burgdorferi.

Nearly complete resonance assignment and the high-resolution NMR structure of the acyl-carrier protein from Borrelia burgdorferi, a target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure-determination pipeline, are reported. This protein was chosen as a potential target for drug-discovery efforts because of its involvement in fatty-acid biosynthesis, an essential metabolic pathway, in bacteria. It was possible to assign >98% of backbone resonances and >92% of side-chain resonances using multidimensional NMR spectroscopy. The NMR structure was determined to a backbone r.m.s.d. of 0.4 Å and contained four α-helices and two 3(10)-helices. A structure-homology search revealed that this protein is highly similar to the acyl-carrier protein from Aquifex aeolicus.

Recent solution structures of RNA and its complexes with drugs, peptides and proteins.

The past two years have seen remarkable progress in the study of RNA structure: the predicted era of RNA structural biology has arrived. Crystallographic structures of the hammerhead ribozyme and of a large subunit of a group I self-splicing intron have begun to reveal the structural basis of RNA enzymatic activity. A remarkable number of structures of small RNAs and of complexes with drugs, peptides and one protein domain have been determined by NMR.

Recent advances in RNA-protein recognition.

The past few years have witnessed remarkable progress in knowledge of the structure and function of RNA-binding proteins and their RNA complexes. X-ray crystallography and NMR spectroscopy have provided structures for all major classes of RNA-binding proteins, both alone and complexed with RNA. New computational and experimental tools have provided unprecedented insight into the molecular basis of RNA recognition.

Structural basis for recognition of the RNA major groove in the tau exon 10 splicing regulatory element by aminoglycoside antibiotics.

Drug-like molecules that bind RNA with sequence selectivity would provide valuable tools to elucidate gene expression pathways and new avenues to the treatment of degenerative and chronic conditions. Efforts at discovering such agents have been hampered, until recently, by the limited knowledge of RNA recognition principles. Several recent structures of aminoglycoside-RNA complexes have begun to reveal the structural basis for RNA-drug recognition. However, the absence of suitable chemical scaffolds known to bind the RNA major groove, where specificity could be provided by the diversity of functional groups exposed on the RNA bases, has represented a major obstacle. Here we report an investigation of the structural basis for recognition of an RNA stem-loop by neomycin, a naturally occurring aminoglycoside antibiotic. We found that neomycin binds the RNA stem-loop that regulates alternative splicing of exon 10 within the gene coding for human tau protein. Mutations within this splicing regulatory element destabilise the RNA structure and cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), an autosomal dominant condition leading to neurodegeneration and death. The three-dimensional structure of the RNA-neomycin complex shows interaction of the drug in the major groove of the short RNA duplex, where familial mutations cluster. Analysis of the structure shows how aminoglycosides and related drugs bind to the RNA major groove, adding to our understanding of the principles of drug-RNA recognition.

A cap for all occasions.

The 5' end of each polymerase II transcript is capped by a methylated guanosine triphosphate. The cap earmarks the mRNA for subsequent processing and nucleocytoplasmic transport, protects the mRNA from degradation and promotes efficient initiation of protein synthesis. The recently solved structures of capping enzymes and cap-protein complexes shed light on how the 5' ends of mRNAs are modified, and reveals the mechanisms by which the cap is recognized and how it functions in a diverse range of processes.

Structure of the P1 helix from group I self-splicing introns.

The upstream cleavage site of group I self-splicing introns is identified by an absolutely conserved U.G base-pair within a double helix. Mutant introns with a wobble C.A substitute are catalytically active, but all other combinations of nucleotides at these positions abolish splicing, suggesting that an unusual RNA structure generated by the wobble pair is recognized by the catalytic intron core. The solution structure of a 20-mer oligonucleotide containing a UUCG tetraloop hairpin and a U.G wobble pair within a double helix was determined by NMR spectroscopy without any assumptions on RNA conformation. Isotopically (15N/13C)-labelled RNA was used to collect an unusually large number of experimental constraints (703 in total, corresponding to approximately 35 constraints per nucleotide) leading to the determination of a structure with very high precision (overall root-mean-square-deviation (rmsd) between 20 converged structures 1.22 A, local rmsd 0.6 A for the tetraloop and 0.85 A for the stem). Analysis of the double helical structure at the conserved U.G wobble pair reveals local distortions from the regular A-form pattern, that may constitute the characteristic feature of U.G wobble pair recognized by the group I intron core and by amino acyl tRNA synthetases. Re-examination of the previously determined tetraloop structure reveals a novel U.G base-pair with a syn guanosine and hydrogen bonding contacts involving both base protons and a sugar 2'-OH. This explains the great stability of RNA UUCG loops when compared with DNA loops of identical sequence, and is one of the first NMR observations of RNA 2'-OH resonances.

How accurately and precisely can RNA structure be determined by NMR?

The great diversity of RNA biological functions has led to widespread interest in RNA structure. Advances in synthetic and spectroscopic techniques have recently allowed the extension of NMR methods of structure determination to RNA structures of significant size and increased biological significance. However, it has not yet been established how accurately and precisely RNA structure can be determined by NMR. The extensive simulations presented here establish credible limits on accuracy and precision of NMR-derived RNA structures and provide quantitative calibrations to evaluate new structures. Synthetic sets of NMR constraints were generated from a crystallographically-derived ribozyme structure. The target structure was then redetermined using approximations and computational protocols derived from our experimental work. The results demonstrate that the structure of RNA molecules of at least 15,000 Da can be determined with precision and accuracy of 1 to 1.5 A, comparable to values obtained for proteins of similar size. Most encouragingly, it is shown that larger, globular and biologically more important RNA structures can be determined with significantly better accuracy and precision than smaller, elongated structures investigated until now.

The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein.

The major packaging signal of human immunodeficiency virus type 1 (HIV-1) RNA has been localised to the region 3' to the major splice donor within the leader sequence. Secondary structural studies for this region of the HIV-1 genome have shown the existence of a stem-loop structure capped by a purine-rich tetraloop. Extensive mapping data presented here lead to the complete characterisation of the structure of the stem-loop, including a new purine-rich internal loop in the lower part of the structure and the previously established GGAG tetraloop at its tip. Biochemical analysis reveals that both internal loop and tetraloop are primary sites for interaction with Gag polyprotein, and that binding of Gag protein leads to a conformational change which alters the RNA structure. NMR spectroscopy has been used to determine the three-dimensional structure of this complete stem-loop structure. The structural analysis reveals a significant difference between the apical part of the stem-loop structure, which adopts a well-defined conformation, and the purine-rich internal loop, which is instead very flexible. In contrast to what is generally observed for internal loop structures in RNA, this region of the encapsidation signal adopts a structure lacking stable interstrand interactions capable of stabilising a unique conformation. We suggest that the stem-loop structure represents a nucleation site for Gag protein binding, and that the protein exploits the flexibility of the internal loop to initiate the unwinding of the structure with successive addition of Gag molecules interacting with the RNA and each other through conserved I (interaction) domains.

The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein.

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is introduced to the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop encoded by the 5' leader sequence found on all HIV-1 mRNAs. We have used multidimensional heteronuclear NMR to determine the structure of the TAR RNA in the presence of the ADP-1 polypeptide, a 37-mer that carries the minimal RNA recognition region of the Tat protein and closely mimics Tat binding specificity. In the presence of a variety of ligands, including ADP-1, related basic peptides and the amino acid derivative argininamide, the bulge region of TAR undergoes a local conformational rearrangement and forms a more stable structure. The structure of TAR in the bound form has been determined from over 1000 NMR-derived constraints. The U23 residue at the 5' end of the bulge is positioned near G26 and A27 in the major groove, rather than stacked on A22 as in the free TAR. U23 and G26 are brought into close proximity by contacts to the guanidinium group and side-chain amide group of a common arginine residue. However, the interaction of this guanidinium group with TAR is not the only source of binding specificity. Besides NOEs to the arginine residue participating in the conformational change, ADP-1 shows additional intermolecular NOEs to TAR, suggesting that there are multiple points of contacts between TAR RNA and residues from the basic and core regions of Tat. These structural results provide important clues towards the identification of small molecular mass and/or peptidomimetic inhibitors of the essential Tat-TAR interaction.

Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding.

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

Changes in side-chain and backbone dynamics identify determinants of specificity in RNA recognition by human U1A protein.

The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein domains, and is found in many proteins involved in recognition of a wide variety of RNAs. Two structures of RNA complexes of human U1A protein have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning how RNP domains discriminate between different RNAs. In this work, we extend the investigation of U1A-RNA recognition by comparing the dynamics of U1A protein both free and in complex with RNA. We have also investigated the trimolecular complex between two U1A proteins and the complete polyadenylation inhibition element to study the effect of RNA-dependent protein-protein interactions on protein conformational flexibility. We report that changes in backbone dynamics upon complex formation identify regions of the protein where conformational exchange processes are quenched in the RNA-bound conformation. Furthermore, amino acids whose side-chains experience significant changes in conformational flexibility coincide with residues particularly important for the specificity of the U1A protein/RNA interaction. This study adds a new dimension to the description of the coordinated changes in structure and dynamics that are critical to define the biological specificity of U1A and other RNP proteins.

Novel techniques in nuclear magnetic resonance for nucleic acids.

Recent techniques for the efficient preparation of isotopically labelled RNA of desired sequence represent a dramatic step forward for NMR of nucleic acids. Three- and four-dimensional NMR experiments greatly facilitate spectral analysis and quantification of structural constraints. Backbone-driven assignment procedures have been introduced to parallel the powerful assignment methods introduced for work with proteins. Additional structural information to complement interproton distances, namely scalar coupling constants defining the backbone conformation, can be obtained using isotopically labelled oligonucleotides. The additional interproton distance and dihedral angle constraints resolved in higher-dimensional spectra will enable the determination of larger DNA and RNA structures and also increase accuracy and precision.

Structure-based and combinatorial search for new RNA-binding drugs.

The discovery by structure-based and combinatorial methods of new RNA-binding drugs presents great opportunities for pharmacological development against drug-resistant bacterial and viral pathogens. A handful of recent RNA structures and more numerous studies of the interaction of combinatorial libraries and oligomeric RNA-binding compounds are providing the foundation for effective RNA-targeted drug discovery programs.

Nonspecific interactions in dye binding to DNA. Influence of alcohols and amides.

The binding of a few drugs (ethidium bromide, propidium diiodide, proflavine and actinomycin D) to DNA has been investigated in aqueous solutions to which cosolvents of different polarity have been added. It is found that both alcohols (less polar than water) and amides (more polar) lower the binding constant according to a linear relationship between the intercalation free energy and cosolvent concentration. The main action of cosolvents cannot be described in terms of electrostatic effects, since they predict much smaller changes in the binding constant than those observed. It appears instead that relevant solvation effects are responsible for the binding strength of the different dyes to DNA. As a general result, it is found that solvation effects largely contribute to the intercalation free energy, thereby weakening the influence of nonspecific interactions at the intercalation site.

Oxidation of Cyclohexane by Molecular Oxygen Photoassisted by meso-Tetraarylporphyrin Iron(III)-Hydroxo Complexes.

The photochemical and photocatalytic properties of iron meso-tetraarylporphyrins bearing an OH(-) axial ligand and different substituents in the beta-positions of the porphyrin ring are reported. Irradiation (lambda = 365 nm) in the absence of dioxygen leads to the reduction of Fe(III) to Fe(II) with the formation of OH(*) radicals. Substituents at the pyrrole beta-positions are found to markedly affect the photoreduction quantum yields. Under aerobic conditions, this photoreaction can induce the subsequent oxidation of cyclohexane to cyclohexanone and cyclohexanol by O(2) itself. The process occurs under mild conditions (22 degrees C; 760 Torr of O(2)) and without the consumption of a reducing agent. The polarity of the solvent and the nature of the porphyrin ring have a remarkable effect on the selectivity of the photooxidation process, likely controlling the cleavage of O-O bonds of possible iron peroxoalkyl intermediates. In particular, in pure cyclohexane, oxidation occurs with the selective formation of cyclohexanone; in contrast, in dichloromethane/cyclohexane mixed solvent, the main oxidation product is cyclohexanol. Phenyl-tert-butylnitrone (pbn) has been found to quench the radical chain autooxidation of the substrate thus increasing the yield of cyclohexanol. This becomes the only oxidation product when iron 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin hydroxide (Fe(III)(TDCPP)(OH)) is used as photocatalyst.

Rapid methods for determination of fluoxetine in pharmaceutical formulations.

Two different analytical methods for the quality control of fluoxetine in commercial formulations have been developed and compared: a spectrofluorimetric method and a capillary zone electrophoretic (CZE) method. The fluorescence emission values were measured at lambda=293 nm when exciting at lambda=230 nm. The CZE method used an uncoated fused-silica capillary and pH 2.5 phosphate buffer as the background electrolyte. The extraction of fluoxetine from the capsules consisted of a simple one-step dissolution with methanol/water, filtration and dilution. Both methods gave satisfactory results in terms of precision; the best results were obtained for the electrophoretic method, with RSD% values always lower than 2.0%. The accuracy was assessed by means of recovery studies, which gave very good results, between 97.5 and 102.6%. Furthermore, both methods also have the advantage of being very rapid.

Spectrophotometric determination of silicate traces in hemodialysis solutions.

Reliable methods for the analysis of silicon are of great importance, because it seems that the silicate anion can reduce aluminum bioavailability in patients undergoing dialysis. Thus, a simple and sensitive spectrophotometric method is described for the determination of silicate traces in dialysis solutions. The method is based on the reaction between silicate ions and excess ammonium molybdate reagent to give a yellow silico-molybdic complex. This complex is then reduced to the heteropoly blue compound by means of ascorbic acid. Absorbance values are measured at 830 nm, and are stable for more than 2 h. A good linearity was obtained up to 300 ng ml(-1) of silicon concentration. The accuracy and the precision of the method were good; relative standard deviation values of 2% intraday and of 3.9% interday for six replicates on 40 ng ml(-1) standard silicate solutions were found. Results of the analysis of some commercial hemodialysis solution samples, obtained by means of the 'standard additions' method, are provided.