Dynamics of protein–water mixtures: insight from combined 1H spin-lattice and spin-spin relaxation studies of myoglobin

Aiming to reveal the dynamical properties of highly concentrated protein-water systems and validating models of motion, ¹H spin-lattice and spin-spin relaxation experiments were performed for myoglobin - H₂O (40 % wt. of myoglobin) mixture versus temperature, from 268 K to 310 K. The spin-lattice relaxation studies were conducted using Fast Field Cycling (FFC) NMR relaxometry, in the frequency range from 10 kHz to 20 MHz. The comprehensive set of relaxation data was interpreted in terms of a superposition of relaxation contributions expressed in terms of Lorentzian spectral densities and corresponding to the time scales of the order of 10^-6 s, 10^-8-10^-7 s and 10^-9-10^-8 s. The model was validated against the ¹H spin-spin relaxation data obtained from Time Domain (TD) NMR experiments at 18.5 MHz. The studies were complemented by ¹H spin-lattice (FFC NMR and TD NMR) and spin-spin (TD NMR) relaxation experiments for myoglobin - D₂O (40 % wt. of myoglobin) mixture. Bi-exponential spin-spin relaxation processes were observed for both myoglobin - H₂O and myoglobin - D₂O mixtures. A thorough comparison of the spin-lattice relaxation and spin-spin relaxation rates (both components) for the H₂O and D₂O containing mixtures led to the conclusion that the fast component of the spin-spin relaxation process originates from the pool of ¹H nuclei of myoglobin and is associated with slow dynamics of the protein, while the slow component of the spin-spin relaxation is a counterpart of the spin-lattice relaxation process (observed by FFC NMR and TD NMR) and they both reflect the dynamics of water molecules strongly bound to myoglobin (for myoglobin - H₂O) and a similar dynamics of myoglobin (for myoglobin - D₂O).