NMR structure determination has become routine for proteins less than 25 kDa (Fig. 2, blue boxes). Techniques, such as TROSY, have been developed and applied to investigate systems of larger molecular weight. The major hindrances to investigating large proteins are (1) spectral overlap and (2) line broadening due to the enhancement of relaxation from the overall tumbling of the molecule. In order to obtain NMR spectra for large proteins, deuteration is required, which results in a significant decrease in the number of traditional restraints required for structure calculations. Spin label probes have been used as paramagnetic relaxation enhancing reagents to obtain structural restraints for protein systems for which NOE data is limited. We aim to determine a standard nitroxide probe(s) (with reduced degrees of freedom) and placement of the probes to maximize the influence on the resolution of the structure.

Figure 2. Flow chart of NMR structure determination. Requirements of large proteins and complexes are added in yellow