MEASUREMENT OF AMIDE PROTON-EXCHANGE RATES AND NOES WITH WATER IN C-13 N-15-ENRICHED CALCINEURIN-B/

A rapid and sensitive 2D approach is presented for measuring amide pro ton exchange rates and the NOE interaction between amide protons and w ater. The approach is applicable to uniformly C-13/N-15-enriched prote ins and can measure magnetization exchange rates in the 0.02 to > 20 s -1 range. The experiments rely on selective excitation of the water re sonance, coupled with purging of underlying H(alpha) resonances, follo wed by NOESY- or ROESY-type transfer to amide protons, which are dispe rsed by the amide N-15 frequencies in an HSQC-type experiment. Two sep arate but interleaved experiments, with and without selective inversio n of the H2O resonance, yield quantitative results. The method is demo nstrated for a sample of the calcium-binding protein calcineurin B. Re sults indicate rapid amide exchange for the five calcineurin B residue s that are analogous to the five rapidly exchanging residues in the 'c entral helix' of the homologous protein calmodulin.