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Quantifying Millisecond Exchange Dynamics in Proteins by CPMG Relaxation Dispersion NMR Using Side-Chain H-1 Probes
University of Toronto.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
University of Toronto.
University of Toronto.
2012 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 6, 3178-3189 p.Article in journal (Refereed) Published
Abstract [en]

A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain H-1 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 {C-13,H-1}-glucose and D2O a significant number of isolated side-chain H-1 spins are generated, eliminating such effects. It thus becomes possible to record H-1 dispersion profiles at the beta positions of Ass, Cys, Ser, His, Phe, Tyr, and Trp as well as the gamma 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 H-1 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 H-1 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.

Place, publisher, year, edition, pages
American Chemical Society , 2012. Vol. 134, no 6, 3178-3189 p.
National Category
Engineering and Technology
URN: urn:nbn:se:liu:diva-76535DOI: 10.1021/ja210711vISI: 000301161500059OAI: diva2:515132
Funding Agencies|National Science Foundation|OISE-0852964|Swedish Research Council||Canadian Institutes of Health Research||Natural Sciences and Engineering Research Council of Canada||Available from: 2012-04-12 Created: 2012-04-11 Last updated: 2012-04-12

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