Strategies for NMR assignment and global fold determinations using perdeuterated proteins

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