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

Abstract

The 55-residue OCRE domains of the splicing factors RBM5 and RBM10 contain 15 tyrosines in compact, globular folds. At 25 °C, all 15 tyrosines show symmetric ¹ H NMR spectra, with averaged signals for the pairs of δ- and ϵ-ring hydrogens. At 4 °C, two tyrosines were identified as showing ¹ H NMR line-broadening due to lowered frequency of the ring-flipping. For the other 13 tyrosine rings, it was not evident, from the ¹ H 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 ¹ H 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 2.0×10⁶ to 1.3×10⁸ s^-1 , and we established an upper limit of <1.0×10⁶ 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 ¹ H NMR line-shape analysis into the nanosecond frequency range.

Keywords: aromatic ring flips; microsecond rate processes; nanosecond rate processes; protein dynamics; tyrosine-rich proteins.