The application of NMR chemical shifts is not limited to structur

The application of NMR chemical shifts is not limited to structural analysis of proteins but they have also been shown to encode information about protein dynamics [20]. The inverse weighted sum of backbone secondary chemical

shifts for Cα, CO, Cβ, N and Hα nuclei defines a so-called Random Coil Index (RCI). Although originally defined for the analysis of globular proteins, applications to IDPs will be feasible given the growing number of experimental studies. Dissolving proteins in anisotropic media GKT137831 cost leads to restricted overall reorientation, thus dipolar coupling interactions no longer average to zero leading to residual dipolar couplings (RDCs) that are experimentally observable in NMR spectra [21].

In IDPs, dynamic averaging of conformations differing in size and shape gives rise to non-zero RDCs. For example, negative 1DNH RDCs are found for segments in which the NH vector is largely oriented perpendicular to the polypeptide chain (extended conformations). Conversely, positive 1DNH values are found for α-helical Raf inhibitor segments [22]. Again, a more sophisticated ensemble approach provides information about specific structural properties such as transient secondary and tertiary structures [23] and [24]. Despite the tremendous success of these applications of RDCs in the past care has to be taken in the case of IDPs and careful control experiments have to be employed to ensure that the conformational ensemble is not significantly perturbed by the anisotropic alignment media. A more comprehensive review of the field is beyond the scope of this perspective article and

can be found elsewhere ([25], and references therein). Undoubtedly the most relevant experimental approach to probe transient long-range contacts in IDPs employs the measurement of paramagnetic relaxation enhancements (PREs) [26]. Since 1H–1H nuclear Overhauser effects (NOEs) Cyclooxygenase (COX) are characterized by pronounced distance dependence conventional NOESY experiments are not sensitive enough to probe distances beyond approximately 6 Å, particularly, as the effective populations of compact sub-states are generally rather small in IDPs. To study paramagnetic relaxation enhancements the protein under investigation is chemically modified by attaching paramagnetic spin labels at defined positions. Typically, the thiol groups of Cys residues (introduced via site-directed mutagenesis) are used to covalently attach the spin label. It has to be noted that the introduction of paramagnetic spin labels into the protein affects both chemical shifts (pseudo contact shifts, PCS) and/or signal intensities via dipolar relaxation between the unpaired electron and the 1HN and 15N nuclei [27]. Depending on the specific spin label used these effects will be different.

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