Small structural ensembles for a 17-nucleotide mimic of the tRNA T psi C-loop via fitting dipolar relaxation rates with the quadratic programming algorithm.

Solution structures are typically average structures determined with the help of nmr-derived distance and torsion angle information. However, when a biomolecule populates significantly different conformations, the average structure might be prone to artifacts, and other refinement strategies are necessary. For example, when experimental restraints are used in molecular dynamics simulations in a time-averaged fashion (MDtar), the experimental structural information does no longer need to be satisfied at each step of the simulation; instead, the whole trajectory must agree with the restraints. However, the resulting structural ensembles are large and not unique and it is not trivial to extract the essential dynamic features for a system. Here we demonstrate that large MDtar ensembles can be simplified substantially by reducing the number of members to just a few on the basis of adjusting the individual probabilities of the members with the PDQPRO program [N. B. Ulyanov et al. Biophysical Journal (1995), Vol. 68, p. 13]. This algorithm finds the global minimum for a search function that represents the best match of a given ensemble with the experimental dipolar interproton relaxation rates. We have applied this strategy to a 17-residue RNA hairpin, whose loop exhibited considerable flexibility evident from nmr data. This 17mer is a mimic of the T psi C-loop of tRNA, where nucleotide 54 is usually a ribosylthymidine. The methylation of U54, which is completely buried in transfer ribonucleic acid, is administered by tRNA (m5 U54)-methyltransferase (RUMT). Since the 17mer is a good substrate for RUMT, we previously concluded that the flexibility of the 17mer's loop is a key to how RUMT gains access to the methylation site [L. J. Yao et al. Journal of Biomolecular NMR (1996) Vol. 9. p. 229]. Application of the PDQPRO algorithm to the previously acquired MDtar trajectories allowed us to reduce the number of conformations from several hundred to one major and five or six minor conformations with individual populations from approximately 5% to approximately 50% without any deterioration in the match with the experimental data. The major conformation exhibits a continuation of A-form helicity through part of the loop, involving C60 and U59. In this and most other conformations the methylation site in U54 is no longer buried.