Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B |
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Authors: | Stephan Grzesiek Ad Bax |
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Institution: | (1) Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892 Bethesda, MD, USA |
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Abstract: | Summary A rapid and sensitive 2D approach is presented for measuring amide proton exchange rates and the NOE interaction between amide protons and water. The approach is applicable to uniformly 13C/15N-enriched proteins and can measure magnetization exchange rates in the 0.02 to >20s–1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying H resonances, followed by NOESY-or ROESY-type transfer to amide protons, which are dispersed by the amide 15N frequencies in an HSQC-type experiment. Two separate but interleaved experiments, with and without selective inversion of the H2O resonance, yield quantitative results. The method is demonstrated for a sample of the calcium-binding protein calcineurin B. Results indicate rapid amide exchange for the five calcineurin B residues that are analogous to the five rapidly exchanging residues in the central helix of the homologous protein calmodulin. |
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Keywords: | Hydrogen exchange NOESY ROESY Water Protein 2D NMR Calcineurin B |
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