OCRE Domains of Splicing Factors RBM5 and RBM10: Tyrosine Ring-Flip Frequencies Determined by Integrated Use of 1H NMR Spectroscopy and Molecular Dynamics Simulations

The 55-residue OCRE domains of the splicing factors RBM5 and RBM10 contain 15 tyrosines in compact, globular folds. At 25 degrees C, all 15 tyrosines show symmetric H-1 NMR spectra, with averaged signals for the pairs of delta- and epsilon-ring hydrogens. At 4 degrees C, two tyrosines were identified as showing H-1 NMR line-broadening due to lowered frequency of the ring-flipping. For the other 13 tyrosine rings, it was not evident, from the H-1 NMR data alone, whether they were either all flipping at high frequencies, or whether slowed flipping went undetected due to small chemical-shift differences between pairs of exchanging ring hydrogen atoms. Here, we integrate H-1 NMR spectroscopy and molecular dynamics (MD) simulations to determine the tyrosine ring-flip frequencies. In the RBM10-OCRE domain, we found that, for 11 of the 15 tyrosines, these frequencies are in the range 2.0x10(6) to 1.3x10(8) s(-1), and we established an upper limit of <1.0x10(6) s(-1) for the remaining four residues. The experimental data and the MD simulation are mutually supportive, and their combined use extends the analysis of aromatic ring-flip events beyond the limitations of routine H-1 NMR line-shape analysis into the nanosecond frequency range.