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

Abstract

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 large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13C/2H isotope labeling and molecular simulations to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. We find that ring flips in ubiquitin are quite conserved in the different crystal forms, even though the intermolecular packing is quite different suggesting that intramolecular influences are more important than intermolecular (packing) effects.

NMR Studies of Aromatic Ring Flips to Probe Conformational Fluctuations in Proteins

“…Potential drawbacks in solid-state NMR include the effects of the dipolar and chemical shift anisotropy relaxation mechanisms, which become very efficient if ring flips occur in the microsecond regime, thereby rendering the signals unobservable. The effect of crystal packing on ring flip dynamics in microcrystalline solid-state NMR samples is of potential concern . The detailed intermolecular interactions of exposed aromatic rings can play significant roles in governing ring flip rate constants, ,, but it remains an open question to what extent crystal packing might influence the dynamics of buried rings; apparent discrepancies between solution-state and solid-state NMR data obtained for the small protein GB1 suggest that crystal packing might play a role also in this case. , Further comparative studies are needed in this area.…”

“…Ring flips can be investigated computationally to augment experimental results from NMR, as was recognized in early investigations . MD simulations provide mechanistic details of the actual ring flip process. − Because ring flips of buried aromatic residues usually are rare events, occurring on time scales of microseconds to milliseconds, various types of accelerated MD are usually needed to achieve sufficient sampling of the barrier crossing and to reach statistically stable results. , Nonaccelerated MD simulations allow for qualitative characterization of ring flips into fast (observed frequently in an MD trajectory) or slow (not or rarely observed) categories. , To date, MD simulations have not been able to reliably reproduce experimentally determined ring flip rate constants, , indicating that NMR data provide valuable benchmarks and further highlighting the need for continued, data-guided development of force fields. MD simulations can also be directly guided by NMR data, either through reweighting of conformational ensembles or introducing restraints. − We foresee that significant insights into the mechanisms of ring flips will be attained by combining MD simulations and NMR data describing ring flip dynamics and activation parameters as well as fast time scale fluctuations within the ground-state basin of the aromatic ring itself and surrounding residues.…”

“…Solid-state NMR experiments offer complementary advantages to studying ring flips because dipolar couplings are averaged by molecular dynamics in such a way that the amplitude and anisotropy of the underlying motion can be determined. ,,,, Furthermore, molecular size is not a limiting factor in solid-state NMR, as long as the analysis is not hampered by spectral overlap due to the increased number of aromatic spins. Potential drawbacks in solid-state NMR include the effects of the dipolar and chemical shift anisotropy relaxation mechanisms, which become very efficient if ring flips occur in the microsecond regime, thereby rendering the signals unobservable.…”

“…By studying ring flips of Phe and Tyr residues in different types of environments, e.g., inside the protein core or on the surface, and as a function of physical parameters, e.g., temperature or pressure, it is possible to extract unique information about this type of dynamic process in terms of its dependence of molecular structure and its thermodynamic activation parameters. Early ground-breaking work by Wagner and Wüthrich revealed great variability in flip rate constants among aromatic rings located in different environments and also provided the first characterization of the activation barriers and activation volumes. − However, despite the long history of studying ring flips, the number of proteins for which ring flip rate constants have been measured quantitatively is still very low. − The scarcity of ring flip data is explained by the special challenges in characterizing ring flip rate constants quantitatively, as compared to measurements of many other types of conformational exchange. In addition, the typically slow time scale of ring flips makes them difficult to sample by MD simulations.…”

“… 10 − 13 However, despite the long history of studying ring flips, the number of proteins for which ring flip rate constants have been measured quantitatively is still very low. 11 − 24 The scarcity of ring flip data is explained by the special challenges in characterizing ring flip rate constants quantitatively, as compared to measurements of many other types of conformational exchange. In addition, the typically slow time scale of ring flips makes them difficult to sample by MD simulations.…”