Protein dynamics information

This approach yields spectral densities. Although it does not require assumptions about the correlation function and therefore is not subjected to the limitations intrinsic to the model-free approach, obtaining information about protein dynamics by this method is no more straightforward, because it involves a similar problem of the physical (protein-relevant) interpretation of the information encoded in the form of SD, and is complicated by the lack of separation of overall and local motions. To characterize protein dynamics in terms of more palpable parameters, the spectral densities will then have to be analyzed in terms of model-free parameters or specific motional models derived e.g. from molecular dynamics simulations. The SD method can be extremely helpful in situations when no assumption about correlation function of the overall motion can be made (e.g. protein interaction and association, anisotropic overall motion, etc. see e.g. Ref. [39] or, for the determination of the 15N CSA tensor from relaxation data, Ref. [27]). 

Which model provides the best representation for local mobility in a particular group remains unclear, as a detailed picture of protein dynamics is yet to be painted. This information is not directly available from NMR measurements that are necessarily limited by the number of experimentally available parameters. Additional knowledge is required in order to translate these experimental data into a reliable motional picture of a protein. At this stage, molecular dynamic simulations could prove extremely valuable, because they can provide complete characterization of atomic motions for all atoms in a molecule and at all instants of the simulated trajectory. This direction becomes particularly promising with the current progress in computational resources, when the length of a simulated trajectory approaches the NMR-relevant time scales [23, 63, 64]. 

Such ambiguity and also the low structural resolution of the method require that the spectroscopic properties of protein fluorophores and their reactions in electronic excited states be thoroughly studied and characterized in simple model systems. Furthermore, the reliability of the results should increase with the inclusion of this additional information into the analysis and with the comparison of the complementary data. Recently, there has been a tendency not only to study certain fluorescence parameters and to establish their correlation with protein dynamics but also to analyze them jointly, to treat the spectroscopic data multiparametrically, and to construct self-consistent models of the dynamic process which take into account these data as a whole. Fluorescence spectroscopy gives a researcher ample opportunities to combine different parameters determined experimentally and to study their interrelationships (Figure 2.1). This opportunity should be exploited to the fullest. 

Bo is the measurement frequency. Rapid exchange between the different fractions is assumed the bulk, water at the protein surface (s) and interior water molecules, buried in the protein and responsible for dispersion (i). In fact, protons from the protein surface exchanging with water lead to dispersion as well and should fall into this category Bulk and s are relevant to extreme narrowing conditions and cannot be separated unless additional data or estimations are available (for instance, an estimation of fg from some knowledge of the protein surface). As far as quadrupolar nuclei are concerned (i.e., and O), dispersion of Rj is relevant of Eqs. (62) and (63) (this evolves according to a Lorentzian function as in Fig. 9) and yield information about the number of water molecules inside the protein and about the protein dynamics (sensed by the buried water molecules). Two important points must be noted about Eqs. (62) and (63). First, the effective correlation time Tc is composed of the protein rotational correlation time and of the residence time iw at the hydration site so that... 

Diffuse scattering, which nearly always occurs during a diffraction experiment however, represents a potentially rich source of dynamic information supplementary to that obtainable from the Bragg reflections. Many protein crystals exhibit diffuse... 

Effective ligand rebinding from the Xel site is only observed when the temperature has risen above a characteristic temperature 180 K (Figure 1.6), which is the temperature where protein dynamics sets in. However, the time-related information is lost in experiments at cryogenic temperatures. Time-resolved crystallography was applied to restore the time scale and observe undisturbed relaxations. 

One of the most widely used tools to assess protein dynamics are different heteronuclear relaxation parameters. These are in intimate connection with internal dynamics on time scales ranging from picoseconds to milliseconds and there are many approaches to extract dynamical information from a wide range of relaxation data (for a thorough review see Ref. 1). Most commonly 15N relaxation is studied, but 13C and 2H relaxation are the prominent tools to characterize side-chain dynamics.70 Earliest applications utilized 15N Ti, T2 relaxation as well as heteronuclear H- N) NOE experiments to characterize N-H bond motions in the protein backbone.71 The vast majority of studies applied the so-called model-free approach to translate relaxation parameters into overall and internal mobility. Its name contrasts earlier methods where explicit motional models of the N-H vector were used, for example diffusion-in-a-cone or two- or three-site jump, etc. Unfortunately, we cannot obtain information about the actual type of motion of the bond. As reconciliation, the model-free approach yields motional parameters that can be interpreted in each of these motional models. There is a well-established protocol to determine the exact combination of parameters to invoke for each bond, starting from the simplest set to the most complex one until the one yielding satisfactory description is reached. The scheme, a manifestation of the principle of Occam s razor is shown in Table l.72... 

From the late 1960 s to the early 1970 s, more direct approaches to the investigation of protein dynamics were intensively developed. Such investigations featured the application of physical methods, such as physical labeling, NMR, optical spectroscopy, fluorescence, differential scanning calorimetry, and X-ray and neutron scattering. The purposeful application of the approaches made it possible to obtain detailed information on the mobility of different parts of protein globules and to compare this mobility with both the functional characteristics and stability of proteins, and with results of the theoretical calculation of protein dynamics. 

Recent single-molecule experimental studies of proteins provide more detailed views of protein motions, and confirm that a wide variety of timescales is involved in, e.g., catalytic action of enzymes [7,14,15,19,33], Of course, molecular dynamics simulations have been used to probe motions in single proteins for many years, and advances in both theory and computational science have made simulations a powerful approach to building theoretical understanding of protein dynamics [1], The recent introduction of accelerated molecular dynamics methods is helpful in this context [11]. Although detailed dynamical information is sacrificed to the enhanced sampling of conformational space in these methods, which have been shown to access conformational fluctuations that are revealed by nuclear magnetic resonance experiments on the millisecond... 

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