Direct measurements of neurosteroid binding to specific sites on GABAA receptors.

BACKGROUND AND PURPOSE: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. EXPERIMENTAL APPROACH: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. KEY RESULTS: Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3ß-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. CONCLUSIONS AND IMPLICATIONS: The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.

Identification and characterization of RNA pentaloop sequence families.

One of the current methods for predicting RNA tertiary structure is fragment-based homology, which predicts tertiary structure from secondary structure. For a successful prediction, this method requires a library of the tertiary structures of small motifs clipped from previously solved RNA 3D structures. Because of the limited number of available tertiary structures, it is not practical to find structures for all sequences of all motifs. Identifying sequence families for motifs can fill the gaps because all sequences within a family are expected to have similar structural features. Currently, a collection of well-characterized sequence families has been identified for tetraloops. Because of their prevalence and biological functions, pentaloop structures should also be well-characterized. In this study, 10 pentaloop sequence families are identified. For each family, the common and distinguishing structural features are highlighted. These sequence families can be used to predict the tertiary structure of pentaloop sequences for which a solved structure is not available.

Predictions and analyses of RNA nearest neighbor parameters for modified nucleotides.

The most popular RNA secondary structure prediction programs utilize free energy (ΔG°37) minimization and rely upon thermodynamic parameters from the nearest neighbor (NN) model. Experimental parameters are derived from a series of optical melting experiments; however, acquiring enough melt data to derive accurate NN parameters with modified base pairs is expensive and time consuming. Given the multitude of known natural modifications and the continuing use and development of unnatural nucleotides, experimentally characterizing all modified NNs is impractical. This dilemma necessitates a computational model that can predict NN thermodynamics where experimental data is scarce or absent. Here, we present a combined molecular dynamics/quantum mechanics protocol that accurately predicts experimental NN ΔG°37 parameters for modified nucleotides with neighboring Watson-Crick base pairs. NN predictions for Watson-Crick and modified base pairs yielded an overall RMSD of 0.32 kcal/mol when compared with experimentally derived parameters. NN predictions involving modified bases without experimental parameters (N6-methyladenosine, 2-aminopurineriboside, and 5-methylcytidine) demonstrated promising agreement with available experimental melt data. This procedure not only yields accurate NN ΔG°37 predictions but also quantifies stacking and hydrogen bonding differences between modified NNs and their canonical counterparts, allowing investigators to identify energetic differences and providing insight into sources of (de)stabilization from nucleotide modifications.

RNA CoSSMos 2.0: an improved searchable database of secondary structure motifs in RNA three-dimensional structures.

The RNA Characterization of Secondary Structure Motifs, RNA CoSSMos, database is a freely accessible online database that allows users to identify secondary structure motifs among RNA 3D structures and explore their structural features. RNA CoSSMos 2.0 now requires two closing base pairs for all RNA loop motifs to create a less redundant database of secondary structures. Furthermore, RNA CoSSMos 2.0 represents an upgraded database with new features that summarize search findings and aid in the search for 3D structural patterns among RNA secondary structure motifs. Previously, users were limited to viewing search results individually, with no built-in tools to compare search results. RNA CoSSMos 2.0 provides two new features, allowing users to summarize, analyze and compare their search result findings. A function has been added to the website that calculates the average and representative structures of the search results. Additionally, users can now view a summary page of their search results that reports percentages of each structural feature found, including sugar pucker, glycosidic linkage, hydrogen bonding patterns and stacking interactions. Other upgrades include a newly embedded NGL structural viewer, the option to download the clipped structure coordinates in *.pdb format and improved NMR structure results. RNA CoSSMos 2.0 is no longer simply a search engine for a structure database; it now has the capability of analyzing, comparing and summarizing search results. Database URL: http://rnacossmos.com.

Identification and Characterization of New RNA Tetraloop Sequence Families.

There is an abundance of RNA sequence information available due to the efforts of sequencing projects. However, current techniques implemented to solve the tertiary structures of RNA, such as NMR and X-ray crystallography, are difficult and time-consuming. Therefore, biophysical techniques are not able to keep pace with the abundance of sequence information available. Because of this, there is a need to develop quick and efficient ways to predict RNA tertiary structure from sequence. One promising approach is to identify structural patterns within previously solved 3D structures and apply these patterns to new sequences. RNA tetraloops are one of the most common naturally occurring secondary structure motifs. Here, we use RNA Characterization of Secondary Structure Motifs (CoSSMos), Dissecting the Spatial Structure of RNA (DSSR), and a bioinformatic approach to search for and characterize tertiary structure patterns among tetraloops. Not surprising, we identified the well-known GNRA and UNCG tetraloops, as well as the previously identified RNYA tetraloop. However, some previously identified characteristics of these families were not observed in this data set, and some new characteristics were identified. In addition, we also identified and characterized three new tetraloop sequence families: YGAR, UGGU, and RMSA. This new structural information sheds light on the tertiary structure of tetraloops and contributes to the efforts of RNA tertiary structure prediction from sequence.

d-electron count, ion-pairing and diagonal twist angles in metallo-bis(dithiolene) complexes.

Electronic structure calculations for late transition metals coordinated by two dithiolene ligands are found to be consistent with existing structures and also predict the geometries of Ni(I) species for which no solid state structures have been reported. Of particular interest are the compounds [M(mnt)2 ]n- (M = Ni, Pd, and Pt with n = 1, 2, 3; M = Cu with n = 2). Calculations have been performed with and without ion-paring with M(diglyme)+ (M = Li, Na, K) and R4 N+ (R = Me, Bu). The diagonal twist angle between two NiS2 planes is found to depend on (i) the metal's d-electron count, spanning from 0° (planar d7 and d8 ), to 42° (d9 ), to 90° (pseudo-tetrahedral d10 ), and (ii) the identity of the ion-paired cations. Calculated ion-pairing energies are functions of the cation size and charge-density, being larger for alkali-metal coordinated diglyme and smaller for tetra-alkyl ammonium cations. © 2016 Wiley Periodicals, Inc.

Predicting the Strength of Anion-π Interactions of Substituted Benzenes: the Development of Anion-π Binding Substituent Constants.

A computational study aimed at accurately predicting the strength of the anion-π binding of substituted benzenes is presented. The anion-π binding energies (Ebind) of 37 substituted benzenes and the parent benzene, with chloride or bromide were investigated at the MP2(full)/6-311++G** level of theory. In addition, energy decomposition analysis was performed on 27 selected chloride-arene complexes via symmetry adapted perturbation theory (SAPT), using the SAPT2+ approach. Initial efforts aimed to correlate the anion-π Ebind values with the sum of the Hammett constants σp (Σσp) or σm (Σσm), as done by others. This proved a decent approach for predicting the binding strength of aromatics with electron-withdrawing substituents. For the Cl--substituted benzene Ebind values, the correlation with the Σσp and Σσm values of aromatics with electron-withdrawing groups had r2 values of 0.89 and 0.87 respectively. For the Br--substituted benzene Ebind values, the correlation with the Σσp and Σσm values of aromatics with electron-withdrawing groups had r2 values of 0.90 and 0.87. However, adding aromatics with electron-donating substituents to the investigation caused the correlation to deteriorate. For the Cl--substituted benzene complexes the correlation between Ebind values and the Hammett constants had r2 = 0.81 for Σσp and r2 = 0.84 for Σσm. For the Br--substituted benzene complexes, the respective r2 values were 0.71 for Σσp and 0.79 for Σσm. The deterioration in correlation upon consideration of substituted benzenes with electron-donating substituents is due to the anion-π binding energies becoming more attractive regardless of what type of substituent is added to the aromatic. A similar trend has been reported for parallel face-to-face substituted benzene-benzene binding. This is certainly counter to what electrostatic arguments would predict for trends in anion-π binding energies, and this discrepancy is further highlighted by the SAPT2+ calculated electrostatic component energies (Eele). The Eele values for the Cl--substituted benzene anion-π complexes are all more binding than the Eele value for the Cl--benzene complex, with the exception of chloride-1,3,5-trimethylbenzene. Again, this is a similar trend to what has been reported for parallel face-to-face substituted benzene-benzene binding. A discussion on this surprising result is presented. In addition, an improved approach to predicting the relative anion-π binding strength of substituted benzene is developed using the results of the SAPT2+ calculations.

RNA CoSSMos: Characterization of Secondary Structure Motifs--a searchable database of secondary structure motifs in RNA three-dimensional structures.

RNA secondary structure is important for designing therapeutics, understanding protein-RNA binding and predicting tertiary structure of RNA. Several databases and downloadable programs exist that specialize in the three-dimensional (3D) structure of RNA, but none focus specifically on secondary structural motifs such as internal, bulge and hairpin loops. The RNA Characterization of Secondary Structure Motifs (RNA CoSSMos) database is a freely accessible and searchable online database and website of 3D characteristics of secondary structure motifs. To create the RNA CoSSMos database, 2156 Protein Data Bank (PDB) files were searched for internal, bulge and hairpin loops, and each loop's structural information, including sugar pucker, glycosidic linkage, hydrogen bonding patterns and stacking interactions, was included in the database. False positives were defined, identified and reclassified or omitted from the database to ensure the most accurate results possible. Users can search via general PDB information, experimental parameters, sequence and specific motif and by specific structural parameters in the subquery page after the initial search. Returned results for each search can be viewed individually or a complete set can be downloaded into a spreadsheet to allow for easy comparison. The RNA CoSSMos database is automatically updated weekly and is available at http://cossmos.slu.edu.

Theoretical study of Cl(-)RG (rare gas) complexes and transport of Cl- through RG (RG = He-Rn).

We present a systematic investigation of the accuracy of the various theories and basis sets that can be applied to study the interaction of Cl(-) ions with Ar atoms. It is conclusively shown that gaseous ion mobility can distinguish among theoretical ion-neutral interaction potentials. Based on the conclusions, high-level ab initio potential energy curves are obtained for all of the Cl(-)-RG (RG = He-Rn) complexes. Spectroscopic constants have been derived from these potentials and are compared to a range of theoretical and experimental data, to which they generally show good agreement. General trends are discussed in comparison to other halogen-rare gas complexes previously studied. The potentials also have been tested by using them to calculate transport coefficients for Cl(-) moving through a bath of RG atoms.

Face-to-face arene-arene binding energies: dominated by dispersion but predicted by electrostatic and dispersion/polarizability substituent constants.

Parallel face-to-face arene-arene complexes between benzene and substituted benzenes have been investigated at the MP2(full)/6-311G** and M05-2X/6-311G** levels of theory. A reasonably good correlation was found between the binding energies and the ∑|σ(m)| values of the substituted aromatics. It is proposed that a substituent |σ(m)| value informs on both the aromatic substituent dispersion/polarizability and the effect the substituent has on the aromatic electrostatics. Supporting this hypothesis, a combination of electrostatic (∑σ(m)) and dispersion/polarizability (∑M(r)) substituent constant terms gives an excellent, and statistically significant, correlation with the benzene-substituted benzene binding energy. Symmetry adapted perturbation theory energy decomposition calculations show the dominant attractive force is dispersion; however, the sum of all nonelectrostatic forces is essentially a constant, while the electrostatic component varies significantly. This explains the importance of including an electrostatic term when predicting benzene-substituted benzene binding energies.

Structural characterization of naturally occurring RNA single mismatches.

RNA is known to be involved in several cellular processes; however, it is only active when it is folded into its correct 3D conformation. The folding, bending and twisting of an RNA molecule is dependent upon the multitude of canonical and non-canonical secondary structure motifs. These motifs contribute to the structural complexity of RNA but also serve important integral biological functions, such as serving as recognition and binding sites for other biomolecules or small ligands. One of the most prevalent types of RNA secondary structure motifs are single mismatches, which occur when two canonical pairs are separated by a single non-canonical pair. To determine sequence-structure relationships and to identify structural patterns, we have systematically located, annotated and compared all available occurrences of the 30 most frequently occurring single mismatch-nearest neighbor sequence combinations found in experimentally determined 3D structures of RNA-containing molecules deposited into the Protein Data Bank. Hydrogen bonding, stacking and interaction of nucleotide edges for the mismatched and nearest neighbor base pairs are described and compared, allowing for the identification of several structural patterns. Such a database and comparison will allow researchers to gain insight into the structural features of unstudied sequences and to quickly look-up studied sequences.