Conformational ensembles of RNA oligonucleotides from integrating NMR and molecular simulations

RNA molecules are key players in numerous cellular processes and are characterized by a complex relationship between structure, dynamics, and function. Despite their apparent simplicity, RNA oligonucleotides are very flexible molecules, and understanding their internal dynamics is particularly challenging using experimental data alone. We show how to reconstruct the conformational ensemble of four RNA tetranucleotides by combining atomistic molecular dynamics simulationswith nuclear magnetic resonance spectroscopy data. The goal is achieved by reweighting simulations using a maximum entropy/Bayesian approach. In this way, we overcome problems of current simulation methods, as well as in interpreting ensemble- and time-averaged experimental data. We determine the populations of different conformational states by considering several nuclear magnetic resonance parameters and point toward properties that are not captured by state-of-the-art molecular force fields. Although our approach is applied on a set of model systems, it is fully general and may be used to study the conformational dynamics of flexible biomolecules and to detect inaccuracies in molecular dynamics force fields

Refinement of molecular dynamics ensembles using experimental data and flexible forward models

A novel method combining maximum entropy principle, the Bayesian-inference of ensembles approach, and the optimization of empirical forward models is presented. Here we focus on the Karplus parameters for RNA systems, which relate the dihedral angles of $\gamma$, $\beta$, and the dihedrals in the sugar ring to the corresponding $^3J$-coupling signal between coupling protons. Extensive molecular simulations are performed on a set of RNA tetramers and hexamers and combined with available nucleic-magnetic-resonance data. Within the new framework, the sampled structural dynamics can be reweighted to match experimental data while the error arising from inaccuracies in the forward models can be corrected simultaneously and consequently does not leak into the reweighted ensemble. Carefully crafted cross-validation procedure and regularization terms enable obtaining transferable Karplus parameters. Our approach identifies the optimal regularization strength and new sets of Karplus parameters balancing good agreement between simulations and experiments with minimal changes to the original ensemble.Comment: Submitted to journal; added zenodo link; replaced fig. 3 with correct on

Folding of the transcription factor Brinker and interactions of the bacterial second messenger c-di-GMP studied by NMR

Nuclear magnetic resonance (NMR) spectroscopy is a technique, which allows the non-invasive investigation of structures, dynamics and interactions of biomolecules. The main goal of this thesis was to elucidate the folding mechanism of the transcription factor Brinker and its implications for DNA recognition as well as the characterization of unfolded protein states by NMR. This constitutes the first part of this thesis. The transcription factor Brinker is a nuclear repressor, which is involved in cellular growth and differentiation. In the absence of DNA, Brinker is completely disordered. However, in the presence of DNA or at low temperatures, the Brinker DNA binding domain (BrkDBD) adopts a well-folded structure. Thus, BrkDBD represents an extreme case of the coupling between binding and folding phenomenon. We have aimed to elucidate this folding mechanism in order to understand its implications for DNA recognition. From our data, it is clear that the BrkDBD folding energy landscape sharply depends on buffer anion type and concentration. We show that folded BrkDBD always adopts the same structure irrespective of the conditions. Our data indicate helical propensity for 3 of the 4 native helices even in unfolded BrkDBD, which may serve as initial contact points for DNA recognition. Resonance broadening due to conformational exchange on the micro- to millisecond time scale between folded and unfolded BrkDBD was analyzed by NMR relaxation dispersion experiments indicating a two-state folding mechanism. Only few residues show a different behavior and these are all located at the DNA binding interface. This local conformational heterogeneity may be important for DNA recognition. Based on these findings, we propose a mechanism of DNA recognition by BrkDBD, where the electrostatics-driven folding is a key component, accelerating the recognition process. In addition, we have analyzed the side-chain chi1-rotamer distribution of urea-denatured ubiquitin and protein G, revealing that individual residues show significant deviations from statistical-coil ensemble averages, indicating local bias towards the folded state. The second part of this thesis describes the quantitative characterization of the intermolecular interactions between monomers of the bacterial second messenger c-di-GMP at physiologically relevant concentrations. C-di-GMP is a bacterial second messenger, involved in many signaling events. Its most important effect is to trigger the transition from motile to sessile bacterial life-styles which plays a major role in biofilm formation. In solution, c-di-GMP has been reported to form several oligomers in the presence of monovalent cations, particularly potassium. However, only monomeric and dimeric c-di-GMP have been observed in complexes with proteins or RNA. We have carried out a detailed kinetic and thermodynamic analysis of c-di-GMP polymorphism in the presence of potassium, which showed that predominantly monomers and only few dimers exist at physiological concentrations. Additionally, we present NOE and ROE structural information on c-di-GMP oligomers, which indicate that these are not entirely all-syn and all-anti as opposed to the literature

The solution structure of the circular trinucleotide cr(GpGpGp) determined by NMR and molecular mechanics calculation.

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum