Measurement of RDCs

Pioneering studies have also been published for weakly aligned protein complexes [61, 62]. Note that the ratio of bound ligand to free ligand must be optimized in order to achieve the desired accuracy when extrapolating the RDCs of the bound state. Since the protein does not interfere with the measurement of RDCs of the ligand, it is recommended to use a high concentration of the former. 

Fig. 10. Measurement of RDCs in a second, different alignment medium can significantly reduce the possible corresponding internuclear vector orientations. Permissible orientations will correspond to the intersection of cones, which are placed according to the respective principal axis systems of ordering.

Recent results have been summarized in a number of articles.14-20 This review in parts is based on ref. 20 and will focus on the measurement and application of RDCs on small- to medium-sized organic molecules after a short introduction into dipolar couplings and the alignment tensor, the most important alignment media for small molecule applications and how to scale their alignment properties in order to get ideal measurement conditions are discussed. A selection of pulse sequences adapted specifically to the measurement of RDCs at natural isotope abundance, and a look at the large variety of applications will complete the overview of the method. Finally, a brief summary and outlook to future perspectives of RDCs will be given. 

The inclusion of average Hamiltonian theory derived pulse sequence building blocks specifically designed for the measurement of RDCs is highly desirable. The homonuclear dipolar decoupling sequences used in ref. 190 lead to the less complex multiplet structures observed in isotropic samples with the chemical shift resolution reduced by a factor of 2-3, depending on the multiple pulse... 

The methods for the measurement of RDCs surveyed in this review are those currently used in the field. It therefore is possible that some already published high-resolution NMR methods provide more efficient alternatives. Further development in this area is expected. 

An introduction of a coexpressed lanthanide-binding tag (LBT) as a method for protein alignment facilitating the measurement of RDCs has been proposed by Wohnert et The LBT chosen by them had the sequence YID-TNNDGWYEGDELLA, and was appended to the terminus of human ubiquitin by standard cloning techniques. 

---