Protein tumbling

P 20-100 Protein tumbling Protein tumbling N, B, B question this interpretation... 

Slowly tumbling large molecules, such as proteins, undergo rapid transverse relaxation, which causes line broadening in the NMR spectrum. This imposes an upper limit on the size of molecules whose structures can be usefully interpreted by NMR. Small molecules tumble at high rates and have much slower relaxation rates, and therefore a sharper well-resolved NMR spectrum. 

For folded proteins, relaxation data are commonly interpreted within the framework of the model-free formalism, in which the dynamics are described by an overall rotational correlation time rm, an internal correlation time xe, and an order parameter. S 2 describing the amplitude of the internal motions (Lipari and Szabo, 1982a,b). Model-free analysis is popular because it describes molecular motions in terms of a set of intuitive physical parameters. However, the underlying assumptions of model-free analysis—that the molecule tumbles with a single isotropic correlation time and that internal motions are very much faster than overall tumbling—are of questionable validity for unfolded or partly folded proteins. Nevertheless, qualitative insights into the dynamics of unfolded states can be obtained by model-free analysis (Alexandrescu and Shortle, 1994 Buck etal., 1996 Farrow etal., 1995a). An extension of the model-free analysis to incorporate a spectral density function that assumes a distribution of correlation times on the nanosecond time scale has recently been reported (Buevich et al., 2001 Buevich and Baum, 1999) and better fits the experimental 15N relaxation data for an unfolded protein than does the conventional model-free approach. 

Chemical shift and line width are related and can be used to describe conformational changes. The 831P value of 5 ppm and the line width of 20 Hz indicate that dianionic 5 phosphate is very tightly bound to the active site and tumbles with the protein. Lower values of chemical shift indicate more loosely bound phosphate. Higher values of the line width indicate the presence of two conformers in equilibrium. If the open and close form undergo slow interconvertion, two signals are observed in the 31P NMR spectra. 

Finally, the last step of the procedure for optimizing experimental conditions is to identify the denaturation temperature of the protein. This step is important because the rotational tumbling rate of a protein increases with temperature, and faster tumbling results in sharper resonance lines. Therefore, the temperature during the NMR experiments should be as high as possible without denaturating the protein. The denaturation temperature can best be determined by either CD-spectroscopy or one-dimensional NMR. 

The overall tumbling of a protein molecule in solution is the dominant source of NH-bond reorientations with respect to the laboratory frame, and hence is the major contribution to 15N relaxation. Adequate treatment of this motion and its separation from the local motion is therefore critical for accurate analysis of protein dynamics in solution [46]. This task is not trivial because (i) the overall and internal dynamics could be coupled (e. g. in the presence of significant segmental motion), and (ii) the anisotropy of the overall rotational diffusion, reflecting the shape of the molecule, which in general case deviates from a perfect sphere, significantly complicates the analysis. Here we assume that the overall and local motions are independent of each other, and thus we will focus on the effect of the rotational overall anisotropy. 

D11/Dj, from 1 to 10. Symbols correspond to synthetic experimental data generated assuming overall tumbling with rc = 5 ns and various degrees of anisotropy as indicated. Model-free parameters typical of restricted local backbone dynamics in protein core, S2=0.87, T oc =20 ps, were used to describe the effect of local motions. The H resonance frequency was set to 600 MHz. The solid lines correspond to the right-hand-side expression in Eq. (10). 

Fig. 16.2 A simplified scheme of the trNOE concept. The ligand L in the free state has negligible cross-relaxation between protons Hi and H2 because of its rapid tumbling motion. Upon binding to the much slower tumbling protein 7 becomes effective and leads to a transfer of magnetization from Hn to H2. Because of the dynamic equilibrium the ligand is released back into solution where it is still in the magnetization state corresponding to the bound form. The same concept is also applicable to trCCR and trRDC (see Sects. 16.4 and 16.5).

Since local motions of flexible protein domains can randomize the emission dipoles in a manner that does not reflect the overall tumbling time of the entire protein, and since there is an apparent lack of fluorophores with the appropriate properties for larger analytes, most FPIAs are useful only for determination of relatively low-molecular-weight analytes. Nevertheless, a few studies of larger-molecular-weight analytes have been reported. A competitive assay for human... 

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