A role for a single-stranded junction in RNA binding and specificity by the Tetrahymena group I ribozyme.

We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.

Implications of molecular heterogeneity for the cooperativity of biological macromolecules.

Cooperativity, a universal property of biological macromolecules, is typically characterized by a Hill slope, which can provide fundamental information about binding sites and interactions. We demonstrate, through simulations and single-molecule FRET (smFRET) experiments, that molecular heterogeneity lowers bulk cooperativity from the intrinsic value for the individual molecules. As heterogeneity is common in smFRET experiments, appreciation of its influence on fundamental measures of cooperativity is critical for deriving accurate molecular models.

Removal of covalent heterogeneity reveals simple folding behavior for P4-P6 RNA.

RNA folding landscapes have been described alternately as simple and as complex. The limited diversity of RNA residues and the ability of RNA to form stable secondary structures prior to adoption of a tertiary structure would appear to simplify folding relative to proteins. Nevertheless, there is considerable evidence for long-lived misfolded RNA states, and these observations have suggested rugged energy landscapes. Recently, single molecule fluorescence resonance energy transfer (smFRET) studies have exposed heterogeneity in many RNAs, consistent with deeply furrowed rugged landscapes. We turned to an RNA of intermediate complexity, the P4-P6 domain from the Tetrahymena group I intron, to address basic questions in RNA folding. P4-P6 exhibited long-lived heterogeneity in smFRET experiments, but the inability to observe exchange in the behavior of individual molecules led us to probe whether there was a non-conformational origin to this heterogeneity. We determined that routine protocols in RNA preparation and purification, including UV shadowing and heat annealing, cause covalent modifications that alter folding behavior. By taking measures to avoid these treatments and by purifying away damaged P4-P6 molecules, we obtained a population of P4-P6 that gave near-uniform behavior in single molecule studies. Thus, the folding landscape of P4-P6 lacks multiple deep furrows that would trap different P4-P6 molecules in different conformations and contrasts with the molecular heterogeneity that has been seen in many smFRET studies of structured RNAs. The simplicity of P4-P6 allowed us to reliably determine the thermodynamic and kinetic effects of metal ions on folding and to now begin to build more detailed models for RNA folding behavior.

Multiple native states reveal persistent ruggedness of an RNA folding landscape.

According to the 'thermodynamic hypothesis', the sequence of a biological macromolecule defines its folded, active (or 'native') structure as a global energy minimum in the folding landscape. However, the enormous complexity of folding landscapes of large macromolecules raises the question of whether there is in fact a unique global minimum corresponding to a unique native conformation or whether there are deep local minima corresponding to alternative active conformations. The folding of many proteins is well described by two-state models, leading to highly simplified representations of protein folding landscapes with a single native conformation. Nevertheless, accumulating experimental evidence suggests a more complex topology of folding landscapes with multiple active conformations that can take seconds or longer to interconvert. Here we demonstrate, using single-molecule experiments, that an RNA enzyme folds into multiple distinct native states that interconvert on a timescale much longer than that of catalysis. These data demonstrate that severe ruggedness of RNA folding landscapes extends into conformational space occupied by native conformations.

Nanomaterials from ionic block copolymers and single-, double-, and triple-tail surfactants.

Block ionomer complexes (BICs) are prepared from anionic block copolymers and cationic surfactants of different structure or from their mixtures. Drastic changes in the morphology and stability of BIC nanoparticles caused by changes in the composition of the surfactant mixture are demonstrated. Single-tail and double-tail surfactants appear to mix within the BIC, resulting in the formation of rather uniform BIC particles. Morphologies of the particles of these mixed BICs are intermediate between those prepared from pure single- and double-tail surfactants. Particles of BIC prepared from mixtures of single- and triple-tail surfactants are heterogeneous, and FRET experiments indicate that surfactant components in these systems are strongly segregated. The results of this study provide important insights into the formation and structure of the BIC and have implications for various applications of the BIC (e.g., nanomedicine), in which precise control of the shape, size, and other properties is needed.

Fluorescence anisotropy study of aqueous dispersions of block ionomer complexes.

Block ionomer complexes (BIC) of "dual hydrophilic" block copolymers containing ionic and nonionic blocks and oppositely charged surfactants spontaneously form colloidal particles of ca. 80 nm in diameter stable in aqueous dispersions at every composition of the mixture. Packing and dynamics of aliphatic groups of the surfactant in BIC were examined by using the quenching-resolved fluorescence anisotropy (QRFA) method with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. The values of the order parameter and rotational relaxation time in the BIC were higher than those in the surfactant micelles. Incorporation of aliphatic alcohols in the BIC decreased the order parameter and increased the rotational relaxation time. The effects on the order parameter were explained by changes in the surfactant aliphatic group conformation to "fill the gaps" induced by insertion of shorter alcohol molecules. The effects on the relaxation time were attributed to a decrease in repulsion of the surfactant headgroups and expulsion of water from the BIC hydrophobic interior as evidenced by the decrease in micropolarity. The results of this study have implications for potential use of the BIC in pharmaceutics and other fields.

Colloidal stability of aqueous dispersions of block ionomer complexes: effects of temperature and salt.

This work characterized colloidal stability of the dispersions, formed by the complexes of poly(ethylene oxide)-b-poly(sodium methacrylate) and hexadecyltrimethylammonium bromide. At room temperature, the dispersion was stabilized by the poly(ethylene oxide) (PEO) chains and did not aggregate for at least several months. Elevation of temperature caused aggregation of the dispersion because of dehydration of the PEO chains. At initial stages (minutes), the aggregation was reversible and the particles spontaneously redispersed once the temperature was decreased. However, it became irreversible at the later stages (hours), probably indicating fusion of the hydrophobic cores of the BIC particles. Addition of elementary salts led to a decrease of the aggregation temperature. The effects of various salts were dependent on the chemical nature of the ions and were consistent with the Hofmeister series. This behavior was discussed in terms of hydration and London (dispersion) interactions between the ions and the PEO.