13 C and 15 N NMR Study of the Hydration Response of T4 Lysozyme and rB-Crystallin Internal Dynamics A. Krushelnitsky,* ,† T. Zinkevich, ‡ N. Mukhametshina, † N. Tarasova, † Y. Gogolev, † O. Gnezdilov, ‡ V. Fedotov, † P. Belton, § and D. Reichert | Kazan Institute of Biochemistry and Biophysics, Kazan, Russia, Kazan Physical-Technical Institute, Kazan, Russia, UniVersity of East Anglia, Norwich, U.K., and Martin-Luther UniVersity Halle-Wittenberg, Halle, Germany ReceiVed: January 13, 2009; ReVised Manuscript ReceiVed: April 23, 2009 The response to hydration of the internal protein dynamics was studied by the means of solid state NMR relaxation and magic angle spinning exchange techniques. Two proteins, lysozyme from bacteriophage T4 and human RB-crystallin were used as exemplars. The relaxation rates R 1 and R 1F of 13 C and 15 N nuclei were measured as a function of a hydration level of the proteins in the range 0-0.6 g of water/g of protein. Both proteins were totally 15 N-enriched with natural 13 C abundance. The relaxation rates were measured for different spectral bands (peaks) that enabled the characterization of the dynamics separately for the backbone, side chains, and CH 3 and NH 3 + groups. The data obtained allowed a comparative analysis of the hydration response of the protein dynamics in different frequency ranges and different sites in the protein for two different proteins and two magnetic nuclei. The most important result is a demonstration of a qualitatively different response to hydration of the internal dynamics in different frequency ranges. The amplitude of the fast (nanosecond time scale) motion gradually increases with increasing hydration, whereas that of the slow (microsecond time scale) motion increases only until the hydration level 0.2-0.3 g of water/g of protein and then shows almost no hydration dependence. The reason for such a difference is discussed in terms of the different physical natures of these two dynamic processes. Backbone and side chain nuclei show the same features of the response of dynamics with hydration despite the fact that the backbone motional amplitudes are much smaller than those of side chains. Although T4 lysozyme and RB-crystallin possess rather different structural and biochemical properties, both proteins show qualitatively very similar hydration responses. In addition to the internal motions, exchange NMR data enabled the identification of one more type of motion in the millisecond to second time scale that appears only at high hydration levels. This motion was attributed to the restricted librations of the protein as a whole. Introduction The importance of studies on water-protein interactions is universally recognized. Water helps proteins to attain the native structure and dynamics which are necessary for their biological function. 1,2 Water also affects the protein functionality by means of direct involvement in biologically relevant processes. 3 It is important to note that water affects not only surface groups but the hydrophobic interior as well. 4 One view is that internal protein diffusive motions are “slaved” to hydration water; 5 however, distinguishing between a “master” and a “slave” in the protein-water interactions is a very controversial issue. 6 The interaction between water and protein is complex. Evidence from systems where the water-to-protein ratio is high suggest that the effects of protein on water dynamics is quite small. 7 Below about 30% water content, the motions of protein and water may be more tightly coupled. 8,9 The available experimental data show that the transition of a protein from a dry state to solution can be divided into a number of steps. A dehydrated protein is characterized by structural distortions arising due to the formation of non-native hydrogen bonding and electrostatic intra- and intermolecular contacts between hydrophilic groups of proteins in the solid state. 4,10,11 Upon increasing the hydration level up to h ∼ 0.1-0.2 (h is defined as g of water/g of protein), these structural distortions practically disappear, since this amount of water is enough to cover hydrophilic surface groups. 12,13 However, the dynamic and enzymatic properties of a protein at this hydration level are still far from those in solution. Full coverage of a protein surface by one hydration layer (h ∼ 0.4) is believed to be enough for almost full restoration of protein properties to those in solution. The heat capacity of a protein-water system does not change upon further increase of hydration; 1 however, there are several experimental indications that changes, although small, in dynamics and enzymatic activity do occur at higher hydration levels. 13-15 The mechanism of the influence of the hydration water on the protein internal dynamics is one of the key factors in understanding the molecular nature of protein biological activity. The notion of water as a lubricant or plasticizer to protein motions is now widely accepted. 2,6 However, it is not clear exactly how these processes are related to dynamic and structural changes. In addition to its fundamental significance, this problem has an important methodical significance. Even soluble proteins are frequently studied in the solid state, since in the solid state one may obtain some structural and dynamic information on proteins which cannot be obtained in solution because of the * Corresponding author. Address: Kazan Institute of Biochemistry and Biophysics, P.O. Box 30, 420111, Kazan, Russia. Phone: +7 (843) 2319037. Fax: +7 (843) 2927745. E-mail: Krushelnitsky@mail.knc.ru. † Kazan Institute of Biochemistry and Biophysics. ‡ Kazan Physical-Technical Institute. § University of East Anglia. | Martin-Luther University Halle-Wittenberg. J. Phys. Chem. B 2009, 113, 10022–10034 10022 10.1021/jp900337x CCC: $40.75  2009 American Chemical Society Published on Web 06/25/2009