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

• Published in: Chembiochem : a European journal of chemical biology Vol. 22; no. 3; pp. 565 - 570
• Main Authors: Martin, Bryan T., Malmstrom, Robert D., Amaro, Rommie E., Wüthrich, Kurt
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley Subscription Services, Inc 02.02.2021
• Subjects: Aromatic compounds; aromatic ring flips; Domains; Frequency ranges; Hydrogen atoms; Magnetic resonance spectroscopy; microsecond rate processes; Molecular dynamics; nanosecond rate processes; NMR; NMR spectroscopy; Nuclear magnetic resonance; protein dynamics; Residues; Simulation; Spectroscopy; Spectrum analysis; Splicing; Splicing factors; Tyrosine; tyrosine-rich proteins
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202000517

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 1H NMR spectra, with averaged signals for the pairs of δ‐ and ϵ‐ring hydrogens. At 4 °C, two tyrosines were identified as showing 1H NMR line‐broadening due to lowered frequency of the ring‐flipping. For the other 13 tyrosine rings, it was not evident, from the 1H 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 1H 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 1H NMR line‐shape analysis into the nanosecond frequency range. Integrated view of protein dynamics: Combined use of 1H NMR spectroscopy and MD simulations of the tyrosine‐rich OCRE domain provides a comprehensive view of the intramolecular dynamics of this protein, with detailed information on the mobility of all tyrosine rings.