Dynamics of Hydrophobic Core Phenylalanine Residues Probed by Solid-State Deuteron NMR

Abstract

We conducted a detailed investigation of the dynamics of two phenylalanine side chains in the hydrophobic core of the villin headpiece subdomain protein (HP36) in the hydrated powder state over the 298–80 K temperature range. Our main tools were static deuteron NMR measurements of longitudinal relaxation and line shapes supplemented with computational modeling. The temperature dependence of the relaxation times reveals the presence of two main mechanisms that can be attributed to the ring-flips, dominating at high temperatures, and small-angle fluctuations, dominating at low temperatures. The relaxation is non-exponential at all temperatures with the extent of non-exponentiality increasing from higher to lower temperatures. This behavior suggests a distribution of conformers with unique values of activation energies. The central values of the activation energies for the ring-flipping motions are among the smallest reported for aromatic residues in peptides and proteins and point to a very mobile hydrophobic core. The analysis of the widths of the distributions, in combination with the earlier results on the dynamics of flanking methyl groups (Vugmeyster et al., J. Phys. Chem. B 2013, 117, 6129–6137), suggests that the hydrophobic core undergoes slow concerted fluctuations. There is a pronounced effect of dehydration on the ring-flipping motions, which shifts the distribution toward more rigid conformers. The cross-over temperature between the regions of dominance of the small-angle fluctuations and ring-flips shifts from 195 K in the hydrated protein to 278 K in the dry one. This result points to the role of solvent in softening the core and highlights aromatic residues as markers of the protein dynamical transitions.

Solvent-Driven Dynamical Crossover in the Phenylalanine Side-Chain from the Hydrophobic Core of Amyloid Fibrils Detected by 2H NMR Relaxation

“…7 Note, that in the original work only the value of T c * was reported, and we calculated here the value of T c for F58 residues in HP36 for consistent comparison. Another difference in the dynamics between the case of the hydrated fibrils and HP36 is that in the latter the central values $\langle E_a^{\mathit{l}arge}\rangle$ are smaller at 21–25 kJ/mol and the widths of the distributions σ l arg e are also much smaller at 2 kJ/mol.…”

“…38 The main mechanism of relaxation is usually considered to be 180° flips of the aromatic ring. 7, 39–49 Of note is the work of Gall and Opella 48 which demonstrated that phenylalanine residues have a distribution of ring-flip rates.…”

“…For example, for phenylalanine rings inside the hydrophobic core of globular villin headpiece subdomain (HP36), it was necessary to employ a two-mode motional model consisting of π -flips and small-angle fluctuations of the phenyl axis. 7 . A similar model has also been employed for phenylene rings of glassy polymers.…”

“…8, 12, 13 One of the distinguishing features in the onset of these biologically relevant anharmonic motions is their dependence on solvation as well as involvement of concerted fluctuations between different groups. 1, 5–7, 10, 13–20 More localized dynamical cross-overs from one motional mode to another do not require hydration and usually occur at lower temperatures. 1–3, 21 …”

“…32–35 We have recently observed a solvent-driven dynamical transition for a phenylalanine residue in the core of globular villin headpiece subdomain (HP36) that did not entirely disappear in the dry protein, but rather showed a transition temperature which was 80° higher. 7 Thus, to address the question of the presence of the dynamical transition in the fibrils it is necessary to probe aromatic residues. F19’s location inside the core (Figure 1), makes it a key target for such studies.…”