Changes in Lysozyme Flexibility upon Mutation Are Frequent, Large and Long-Ranged

We investigate changes in human c-type lysozyme flexibility upon mutation via a Distance Constraint Model, which gives a statistical mechanical treatment of network rigidity. Specifically, two dynamical metrics are tracked. Changes in flexibility index quantify differences within backbone flexibility, whereas changes in the cooperativity correlation quantify differences within pairwise mechanical couplings. Regardless of metric, the same general conclusions are drawn. That is, small structural perturbations introduced by single point mutations have a frequent and pronounced affect on lysozyme flexibility that can extend over long distances. Specifically, an appreciable change occurs in backbone flexibility for 48% of the residues, and a change in cooperativity occurs in 42% of residue pairs. The average distance from mutation to a site with a change in flexibility is 17–20 Å. Interestingly, the frequency and scale of the changes within single point mutant structures are generally larger than those observed in the hen egg white lysozyme (HEWL) ortholog, which shares 61% sequence identity with human lysozyme. For example, point mutations often lead to substantial flexibility increases within the β-subdomain, which is consistent with experimental results indicating that it is the nucleation site for amyloid formation. However, β-subdomain flexibility within the human and HEWL orthologs is more similar despite the lowered sequence identity. These results suggest compensating mutations in HEWL reestablish desired properties

NMR and dielectric spectroscopy investigation of protein dynamical structure

The general approach to globular proteins dynamical structure investigation by NMR and time domain dielectric spectroscopy (TDDS) is presented. The information on macromolecular dynamical behavior in the case of these two methods is obtained in terms of correlation function and its parameters of atom motions. The interpretation of experimental results in the present work will be carried out in the framework of model-free approach which is common both for magnetic and dielectric relaxation. The lysozyme pH-dependence investigation is presented as an example

Investigation of molecular motions and interprotein interactions in solutions by NMR and TDDS.

Non-selective NMR relaxation of protein and water protons at various resonance frequencies as well as Time Domain Dielectric Spectroscopy (TDDS) were applied to study the molecular motions in lysozyme and myoglobin solutions. It was found that the correlation function of the protein motion defined by means of all these methods can be presented as a sum of three components having substantially different correlation times. Both NMR and TDDS experimental data were treated on the basis of approach according to which these components of the correlation function correspond to three different kinds of protein motion, namely 1) internal local motion, 2) anisotropic rotational Brownian diffusion and 3) translational Brownian diffusion. According to the hypothesis proposed earlier we suppose that the reason of anisotropy of protein rotation and possibility to detect experimentally the slowest motion (translational diffusion) is the mutual interprotein electrostatic steering. The qualitative consistency between parameters of correlation function obtained from NMR and TDDS and their concentration dependence confirm the validity of the qualitative model of the interprotein electrostatic interactions

13C and15N NMR Study of the Hydration Response of T4 Lysozyme and αB-Crystallin Internal Dynamics

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 aB-crystallin were used as exemplars. The relaxation rates R1 and R1? of 13C and 15N 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 15N-enriched with natural 13C 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 CH3 and NH3+ 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 aB-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