Quantifying Millisecond Exchange Dynamics in Proteins by CPMG Relaxation Dispersion NMR Using Side-Chain (1)H Probes.
 

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Quantifying Millisecond Exchange Dynamics in Proteins by CPMG Relaxation Dispersion NMR Using Side-Chain (1)H Probes.

Journal of the American Chemical Society (2012). Volume: 134, Issue: 6. Pages: 3178-3189. Alexandar L Hansen, Patrik Lundström, Algirdas Velyvis, Lewis E Kay et al.

A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain (1)H positions in proteins. Such experiments are not possible in a fully protonated molecule because of magnetization evolution from homonuclear scalar couplings that interferes with the extraction of accurate transverse relaxation rates. It is shown, however, that by using a labeling strategy whereby proteins are produced using {(13)C,(1)H}-glucose and D(2)O a significant number of 'isolated' side-chain (1)H spins are generated, eliminating such effects. It thus becomes possible to record (1)H dispersion profiles at the ? positions of Asx, Cys, Ser, His, Phe, Tyr, and Trp as well as the ? positions of Glx, in addition to the methyl side-chain moieties. This brings the total of amino acid side-chain positions that can be simultaneously probed using a single (1)H dispersion experiment to 16. The utility of the approach is demonstrated with an application to the four-helix bundle colicin E7 immunity protein, Im7, which folds via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. The extracted (1)H chemical shift differences at side-chain positions provide valuable restraints in structural studies of invisible, excited states, complementing backbone chemical shifts that are available from existing relaxation dispersion experiments.

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