NMR and MD studies of the temperature-dependent dynamics of RNA YNMG-tetraloops

Abstract

In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.

Structural fidelity and NMR relaxation analysis in a prototype RNA hairpin

“…The alignment tensor parameters were obtained by fitting RDCs to the initial NMR structure followed by superimposing individual conformations obtained from simulation to this initial NMR structure and using the five-elements of the alignment tensor to calculate the ensemble averaged RDCsenglish. Overall, our calculations show that RDC rms deviation from experiment for simulations using the ff10 force field are 3.4 Hz which is larger (Duchardt et al 2004;Vallurupalli and Kay 2005;Ferner et al 2008;Nozinovic et al 2010a,b). The temperature in this case was 298 K and the final KCl concentration 32 mM.…”

“…A wide variety of NMR relaxation studies have been carried out using the RNA UUCG hairpin analyzed here (Duchardt and Schwalbe 2005;Vallurupalli and Kay 2005;Rinnenthal et al 2007;Ferner et al 2008;Rinnenthal et al 2009;Nozinovic et al 2010b). This offers an unique opportunity to test the ability of current force fields to reproduce such data.…”

“…We summarize these data in Table 3 together with estimates from simulation and the hydroNMR program that is often used to estimate diffusion tensors from structure (Garcia De La Torre et al 2000). Recently, some of the NMR relaxation rates used here as reference for EC2 case (Duchardt and Schwalbe 2005;Ferner et al 2008) have been refitted simultaneously to yield similar overall tumbling times, but significantly larger anisotropies (Berlin et al 2013) and no information on the order parameters.…”

“…The only region that distinguishes between the substates is the G9-G10 linkage. (Ferner et al 2008) obtained from relaxation data of C8H8,C6H6, C2H2,C1 ′ H1 ′ directions.…”

Free Energy Landscape of GAGA and UUCG RNA Tetraloops

“…Their small size, together with the abundance of experimental data available, made these systems primary targets for atomistic molecular dynamics (MD) simulation studies. 2 While simulations initialized in the vicinity of the native state are stable on short time-scales under a variety of simulation conditions, [3][4][5][6][7][8] more recent works strongly suggest that these systems are not correctly modeled by the current Amber force field. [9][10][11][12][13][14] Although different improvements have been proposed, 15 there is growing evidence that none of the available corrections are able to capture the crucial non-canonical interactions present in these tetraloops.…”