Strategies for NMR assignment and global fold determinations using perdeuterated proteins

Abstract

This chapter discusses the benefits of using uniform high-level deuteration to inhibit relaxation processes. This facilitates assignment of larger proteins for structural studies and enables, via edited NOESY experiments, the determination of medium- to long-range distance constraints important in establishing the tertiary organization or global fold of proteins. The proteins studied in the chapter are human carbonic anhydrase II (HCA II), a 29 kDa metalloenzyme, and a 12 kDa core packing mutant of thioredoxin (L78K-TRX), for which the motional dynamics have been characterized. The protein HCA II have been successfully 13C, 15N, and 2H- labeled and the significant advantages in signal-to-noise ratios for heteronuclear nuclear magnetic resonance (NMR) experiments have been demonstrated compared to a fully protonated 13C/15N protein. Using this protein, a general strategy has also been developed for the complete mainchain, as well as carbon and NH x sidechain assignments of perdeuterated proteins. In addition, for both HCA II and L78K-TRX, 3D and 4D 15N/15N-separated NOESY data have been obtained, which show anticipated long range interactions from which distance constraints can be derived. These are currently being evaluated in establishing the global folding patterns for these proteins and the initial results for L78K-TRX are shown in the chapter that confirms the importance and utility of these data in establishing tertiary organization. The rapid determination of protein global folds can enhance the comparison of mutant proteins with their wild-type counterparts and can significantly speed up efforts in drug discovery. In addition, the global fold may subsequently be utilized in, more detailed, structural studies by helping to resolve ambiguities in 4D 13C/13C-separated and 13C/15N-separated NOESY data. These studies indicate that perdeuteration can be achieved in proteins expressed in several different E. coli strains by growing selected cells in D 2 O media. Complete deuteration provides significant signal-to-noise enhancement in heteronuclear NMR assignment and structure determination experiments that use the amide proton for detection.