Dynamics of the hydrate and amide groups of crystalline ribonuclease and lysozyme

Abstract

We have used 2H magnetic resonance to study orientational ordering and dynamics of the hydrate and exchangeable hydrogens of crystalline RNase and lysozyme. In these proteins, a predominant fraction of the hydrate deuterons have negligible quadrupole coupling indicating that the solvate is orientationally disordered at room temperature. This observation is reconciled with the crystallographic observation of a substantial fraction of the water being “ordered” by proposing that water molecules readily move among most of the solvent locations including those sites identified in the X-ray structures. Support for this proposition is based on the observation that spin relaxation of the solvate is characterized by a single relaxation time indicating that the solvate deuterons are exchange averaged in the NMR experiment. With regard to the amide deuterons, the residual quadrupole coupling shows that the peptide planes librate with arms amplitude of 12−15° (287 K). Compared to a crystalline peptide ( T 1 ⪢ 100 s) or solid anhydrous poly-γ-benzyl-L-glutamate ( T 1 ≈ 1 s), spin relaxation of the amide deuterons in these hydrated proteins ( T 1 ≈ 0.2 s) is very efficient. Relaxation results are interpreted with a damped Langevin oscillator model and indicate that reorientation of the protein backbone has a distinctly stochastic nature as opposed to that of undamped solid-like oscillatory modes. Furthermore, while the hydrate is important in damping or plasticizing the backbone dynamics, it is apparently not entirely responsible for this effect.