Sequence-dependent conformational differences of small RNAs revealed by native gel electrophoresis.

In this study, we use native polyacrylamide gel electrophoresis and one-dimensional NMR spectroscopy to analyze small RNA hairpins containing a UUCG tetraloop. The aggregation state of one RNA 16-mer (5'-CGGCUUCGGUCGACCA-3') in the presence of Mg(2+) was confirmed by laser light scattering. Although it is widely known in the RNA field that some RNAs tend to aggregate, especially when present at high concentrations, the sequence elements responsible for this effect are rarely identified. In this work, we show that Mg(2+)-induced aggregation of the 16-mer RNA hairpin is sensitive to the presence of the 3'-terminal base and a specific 2'-hydroxyl group. Our study highlights the fact that even small changes in a particular RNA sequence can increase its tendency to undergo Mg(2+)-dependent aggregation in an unpredictable manner. Our analysis also shows that native gel electrophoresis is a sensitive probe of RNA conformation with the capability to detect differences apparently caused by subtle base stacking effects at the ends of helices.

NMR investigation and secondary structure of domains I and II of rat brain calbindin D28k (1-93).

Calbindin D28k, a member of the troponin C superfamily of calcium-binding proteins, contains six putative EF hand domains but binds only four calcium-atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high-affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I-II (residues 1-93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the 1H, 13C and 15N resonances. The secondary structure of the protein was deduced from the size of the 3JHN-Halpha coupling constants, the chemical shift indices of 1Etaalpha, 13Calpha, 13C' and 13Cbeta nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major alpha-helices are identified: Ala13-Phe23, Gly33-Ala50, Leu54-Asp63, Val76-Leu90, while residues Ala2-Leu6 form a fifth, flexible helical segment. Two short beta-strands (Tyr30-Glu32, Lys72-Gly74) are found preceding helices B and D and are arranged in an anti-parallel interaction. Based on these data a structural model of calb I-II was constructed that shows that the construct adopts a tertiary structure related to other well-described calcium-binding proteins of the EF-hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size-exclusion chromatography and analytical ultra-centrifugation studies.

Structural model of a cyclic dynorphin A analog bound to dodecylphosphocholine micelles by NMR and restrained molecular dynamics.

The compound c[Cys5,11]dynorphin A-(1-11)-NH2, 1, is a cyclic dynorphin A analog that shows similar selectivity and potency at the kappa-opioid receptor when compared to the native form of the peptide in central nervous system assays. Previous molecular mechanics calculations have shown that the ring portion of the isoform that is trans about the Arg9-Pro10 omega bond contains either a beta-turn from residues Arg6 to Arg9 or an alpha-helical conformation. Our results from solution state NMR indicate that the compound exhibits cis-trans isomerism about the Arg9-Pro10 omega bond in both aqueous solution and when bound to dodecylphosphocholine micelles. Restrained molecular dynamics calculations show that the cis isoform of the peptide contains a type III beta-turn from residues Arg7 to Pro10. Similar calculations on the trans isoform show it to contain a beta-turn from residues Cys5 and Arg8. In this report we describe the generation of three-dimensional models from NMR data for the ring portions of both the cis and trans isoforms of 1 bound to dodecylphosphocholine micelles. Comparison with other dynorphin A structural information indicates that both the cis and trans isoforms of the peptide may be active as kappa-opioid agonists.

Role of tryptophan-14 in the interaction of dynorphin A(1-17) with micelles.

Fluorescence spectroscopy has been used to examine the interaction between the opioid peptide dynorphin A(1-17) (dynorphin) and dodecylphosphocholine (DPC) micelles. Fluorescence emission spectra as a function of added lipid indicate insertion of the Trp14 side chain into the hydrophobic portion of the micelle, supporting NMR results from this laboratory. A model of interaction with micelles consistent with the fluorescence results and earlier NMR results is proposed. The critical micelle concentration in the presence of peptide was also determined, and is discussed in the context of relevance to both NMR spectroscopy and peptide-lipid interactions.

NMR and structural model of dynorphin A (1-17) bound to dodecylphosphocholine micelles.

Dynorphin A (1-17) (dynorphin) acts preferentially and with high affinity at the kappa-opioid receptor, for which it is the natural, endogenous ligand. Interest in designing new ligands to interact at the kappa-opioid receptor is based in part on the desire to circumvent some of the problems associated with mu-opioid ligands such as morphine. The high-resolution structure of dynorphin in an environment which closely resembles its environment in vivo could be considered as an important lead for new drugs. The interactions that occur between dynorphin and a model membrane are potentially important, as peptide hormone activity is thought to be mediated by interactions with the cell membrane. Therefore, we have determined the high-resolution structures of dynorphin in a model membrane. Results from our laboratory have shown the existence of an alpha-helical region in dynorphin from residues Gly3 through Arg9 when bound to perdeuterated dodecylphosphocholine (DPC) micelles. In this report we show that dynorphin is bound to DPC micelles and describe a family of dynorphin structures that is alpha-helical from residues Gly3 through Pro10 and that contains a beta-turn from residues Trp14 through Gln17. A model of interaction with the micelle is also reported and is discussed in the context of hormone action in vivo. The structures were determined with 1D and 2D nuclear magnetic resonance spectroscopy, distance geometry in dihedral angle space, and restrained molecular dynamics simulations.

The use of dodecylphosphocholine micelles in solution NMR.

Dodecylphosphocholine (DPC) micelles are useful as a model membrane system for solution NMR. Several new observations on dodecylphosphocholine micelles and their interactions with opioid peptides are described. The optimal lipid concentration has been investigated for small peptide NMR studies in DPC micelles for two opioid peptides, a 5-mer and a 17-mer. In contrast to reports in the literature, identical 2D spectra have been observed at low and high lipid concentrations. The chemical shift of resolved peptide proton resonances has been followed as a function of added lipid and indicates that there are changes in the chemical shifts above the critical micelle concentration and up to a ratio of 7:1 (lipid:peptide) for the 17-mer, and 9.6:1 for the 5-mer. These results suggest that conformational changes occur in the peptide significantly above the critical micelle concentration, up to a lipid:peptide ratio which is dependent upon the peptide, here ranging from 7:1 to 9.6:1. To address the stoichiometry more directly, the diffusion coefficients of the lipid alone and the lipid with peptide have been measured using pulsed-field gradient spin-echo NMR experiments. These data have been used to calculate the hydrodynamic radius and the aggregation number of the micelle with and without peptide and show that the aggregation number of the peptide-lipid complex increases at high lipid concentrations without a concomitant change in the peptide conformation. Last, several protonated impurities have been observed in the commercial preparation of DPC which resonate in the amide proton region of the NMR spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Hairpin opening by single-strand-specific nucleases.

DNA molecules with covalently sealed (hairpin) ends are probable intermediates in V(D)J recombination. According to current models hairpin ends are opened to produce short single-stranded extensions that are thought to be precursors of a particular type of extra nucleotides, termed P nucleotides, which are frequently present at recombination junctions. Nothing is known about the activities responsible for hairpin opening. We have used two single-strand-specific nucleases to explore the effects of loop sequence on the hairpin opening reaction. Here we show that a variety of hairpin ends are opened by P1 nuclease and mung bean nuclease (MBN) to leave short, 1-2 nt single-stranded extensions. Analysis of 22 different hairpin sequences demonstrates that the terminal 4 nt of the hairpin loop strongly influence the sites of cleavage. Correlation of the nuclease digestion patterns with structural (NMR) data for some of the hairpin loops studied here provides new insights into the structural features recognized by these enzymes.

1H NMR of 5'CGCGTATATACGCG3', a duplex and a four-membered loop.

The deoxyribonucleotide 5'CGCGTATATACGCG3' was synthesized and studied by NMR methods. This short, fully palindromic duplex also forms a hairpin under certain conditions described within. As such, it is considered to be a model for cruciform formation. We show that this sequence forms a four-membered loop and a duplex in solution. The duplex is shown to be a normal, B-DNA like helix, while the hairpin is shown to be a four-membered ATAT loop.