Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B.
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 > 20 s-1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying H alpha 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.
Grzesiek
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Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Bax
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D000577:Amides / Q000737:chemistry*; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D011522:Protons; D014867:Water
D000577:Amides; D002148:Calmodulin-Binding Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011522:Protons; D014867:Water; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases