Proteins Studied by NMR
Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for the study of the structure, dynamics, activity, and folding of proteins in solution. Peptides and small proteins ( < 10 kDa) can be studied in detail using 1 H NMR and two-dimensional methods including COSY (correlation spectroscopy) and TOCSY (total correlation spectroscopy), which provide 'through-bond' correlations, and NOESY (nuclear Overhauser effect spectroscopy), which provides 'through-space' information. For larger proteins (10-30 kDa), isotope labeling with 15 N and 13 C is generally required. Resonance assignments are obtained using three-dimensional 15 N-edited TOCSY- and NOESY-HSQC or 1 H- 13 C- 15 N triple-resonance experiments. For proteins larger than ∼30 kDa, high levels of deuteration, in addition to 15 N/ 13 C labeling, and the application of TROSY (transverse relaxation-optimized spectroscopy)-based triple-resonance experiments are required. Once the spectrum has been assigned, structural information is obtained from NOEs, scalar coupling constants, chemical shifts, residual dipolar couplings, and amide exchange data. The restraints derived from these NMR parameters are used as inputs to distance geometry, simulated annealing, and molecular dynamics calculations to derive a three-dimensional structure of the protein molecule in solution. NMR relaxation methods can also be used to describe the dynamics of the polypeptide chain at the level of individual residues. © 2010 Elsevier Ltd All rights reserved.