Backbone motions in a crystalline protein from field-dependent 2H-NMR relaxation and line-shape analysis

Abstract

We have used 2H-nmr to study backbone dynamics of the 2H-labeled, slowly exchanging amide sites of fully hydrated, crystalline hen egg white lysozyme. Order parameters are determined from the residual quadrupole coupling and values increase from S2 = 0.85 at 290 K to S2 = 0.94 at 200 K. Dynamical rates are determined from spin-lattice relaxation at three nmr frequencies (38.8, 61.5, and 76.7 MHz). The approach used here is thus distinct from solution nmr studies where dynamical amplitudes and rates are both determined from relaxation measurements. At temperatures below 250 K, relaxation is independent of the nmr frequency indicating that backbone motions are fast compared to the nmr frequencies. However, as the temperature is increased above 250 K, relaxation is significantly more efficient at the lowest frequency, which shows, in addition, the presence of motions that are slow compared to the nmr frequencies. Using the values of S2 determined from the residual quadrupole coupling and a model-free relaxation formalism that allows for fast and slow internal motions, we conclude that these slow motions have correlation times in the range of 0.1 to 1.0 microsecond and are effectively frozen out at 250 K where fast motions of the amide planes with approximately 15 ps effective correlation times and 9 degrees rms amplitudes dominate relaxation. The fast internal motions increase slightly in amplitude as the temperature rises toward 290 K, but the correlation time, as is also observed in solution nmr studies of RNase H, is approximately constant. These findings are consistent with hypotheses of dynamic glass transitions in hydrated proteins arising from temperature-dependent damping of harmonic modes of motion above the transition point.

Probing temperature- and solvent-dependent protein dynamics using terahertz time-domain spectroscopy

“…The transition that we measured in HEWL crystals occurred at a lower temperature than the typically reported dynamical transition for proteins (180-250 K;Bajaj et al, 2008;Chong et al, 2001;Ferrand et al, 1993;Mack et al, 2000;Doster et al, 1989) but is very close to the HEWL crystal true glass transition, T g , measured by differential scanning calorimetry of HEWL crystals at 150 K (Miyazaki et al, 2000). Neutron scattering studies of HEWL crystals do not include measurements at this temperature (Bon et al, 2002).…”

“…A sharp change in hx 2 i at a given temperature is an indication that a change in the dynamical behaviour of the sample has occurred. Such sharp changes in hx 2 i have been observed in a variety of hydrated proteins (Chong et al, 2001;Ferrand et al, 1993;Mack et al, 2000), polypeptides (Bajaj et al, 2008) and amino acids (Schiró et al, 2011) at temperatures between 180 and 240 K using Mö ssbauer spectroscopy, nuclear magnetic resonance (NMR) and neutron scattering experiments (Bajaj et al, 2008;Chong et al, 2001;Ferrand et al, 1993;Mack et al, 2000), and at temperatures between 180 and 240 K using THz-TDS (Chen et al, 2005;He et al, 2008). It is important to note that the temperature-dependent changes seen in these studies are not attributed to a true glass transition but rather to a change in the dynamical behaviour of the protein molecules within the sample, with similarities to the changes in material properties of liquids when they form a glass.…”

Internal protein dynamics on ps to μs timescales as studied by multi-frequency 15N solid-state NMR relaxation

“…S6-S8) qualitatively provide a very similar picture: fast and intermediate motions peaks separated by a gap and a sharp decrease of the motional amplitude within the timescale range 10 -6 -10 -7 s. Thus, the features we observe are model-independent. The fact that the amplitude of the slow motion of the protein backbone is much smaller than that of the fast motion has been noticed well before (Mack et al 2000). However, our data clearly indicate that the tendency of decreasing amplitude with increasing time scale of the motion is pronouncedly non-monotonic, there are ''ups'' and ''downs'' on this dependence which is a result of the complex nature of the backbone mobility.…”

Probing Protein Dynamics Using Multifield Variable Temperature NMR Relaxation and Molecular Dynamics Simulation

“…Recently, solid-state NMR methods have been developed to an extent which enables the study of site-specific 13 C, 15 N, and 1 H relaxation rates in proteins . Notably, Lewandowski et al presented a solid-state NMR approach to study the hierarchy of protein motions.…”