Several different approaches have been proposed to increase the protein size limit in solution NMR spectroscopy. Heteronuclear triple resonance experiments, enabled through the isotopic enrichment of carbon and nitrogen atoms in protein samples, have rapidly extended this size limit from 10 to 30 kDa during the last decade. The 15N/13C-enrichment, combined with perdeuteration, allows the assignment of proteins in the size regime of 40 to 50 kDa. Very recently, transverse relaxation optimized spectroscopy has pushed the size limit even further, and as a result, backbone resonance assignment has been successfully obtained for a 110 kDa octamer (Salzmann et al. 2000). However, protein structure determination is still a very time-consuming procedure, especially in the case of assignment of NOEs. On the other hand, the use of perdeuterated samples suppresses a major part of the information available from NOESY spectra for global fold determination, ultimately leading to loose models. It is therefore inevitable that special means are needed to speed up global fold recognition and to complement long-range distance information lost through the perdeuteration procedure. Use of residual dipolar couplings serves as a powerful solution to both problems.
It has been shown that relatively precise structures can be determined by utilizing residual dipolar couplings with the aid of a small quantity of NOE information (Clore et al. 1998b; Mueller et al. 2000). Thus, the aim should be placed on the structure determination of highly perdeuterated or site-specifically protonated proteins, where the transverse relaxation times of 15N/13Cα spins can be rather long when the TROSY approach is used. Thus, the protein backbone assignment can be retrieved using spectra with high sensitivity and resolution. The measurement of dipolar couplings can be accomplished in a relatively short time compared with traditional NOE analysis. The measurement of changes in the residual dipolar couplings supplemented with chemical shift changes due to ligand binding can provide unique and valuable insight into the structural biology of protein and protein complexes. The directional information, available through several residual dipolar couplings, can be obtained as soon as the protein backbone assignment procedure is accomplished. The experiments presented can supply up to nine residual dipolar couplings in protonated or perdeuterated proteins. Two-dimensional spin-state-selective 15N, 1H -detected experiments can be very handy in structure activity relationship (SAR) by NMR studies (Shuker et al. 1996; Hajduk et al. 1997). Thus, the wealth of information available through residual dipolar couplings can be used concomitantly with chemical shift changes as an indicator of conformational changes induced by ligand binding. The spin-state-selective 15N, 1H -detected experiments are the most suitable ones for large proteins, owing to the inherently good dispersion of the 15N, 1H correlation map. Furthermore, selection of the most slowly relaxing (TROSY) component, especially in perdeuterated proteins, improves the sensitivity of the 15N, 1H -detected experiments considerably. For the largest or highly helical proteins, it may be necessary to record three-dimensional HNCO-type experiments, combined with spin-state-selective subspectral editing and TROSY selection. This approach also provides minimal resonance overlap in larger α-helical proteins, which otherwise often exhibit overcrowded spectra.
Development in experimental methodology and new labeling techniques, as well as technical advancements in NMR instrumentation, have not only increased the protein size limit but have also enabled more convenient and reliable structure determination of smaller proteins. The proposed IM-HSQC experiment enables precise and efficient determination of the structurally most important three-bond J-coupling between 1HN and 1Hα spins from 15N-HSQC-type spectra. It is also applicable to protein samples with minimum isotope labeling, i.e. 15N-enrichment.