Aromatic ring flips in differently packed ubiquitin protein crystals from MAS NMR and MD

Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein's hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13 C/2H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear are remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10-20 ns time scale, and Phe45 is broadened in all crystals, presumably due to microsecond motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects. Competing Interest Statement The authors have declared no competing interest. Footnotes * More analyses of MD simulations (Figure 6, Figure S2 and S3, S5). Tables with experimental and fitted data (Tables S1 and S2). More extensive discussion.