SATURATION-TRANSFER OF EXCHANGEABLE PROTONS IN H-1-DECOUPLED N-15 INEPT SPECTRA IN WATER - APPLICATION TO THE MEASUREMENT OF HYDROGEN-EXCHANGE RATES IN AMIDES AND PROTEINS

Polarization-transfer experiments such as INEPT or DEPT improve the sensitivity of nuclei of low gyro magnetic ratio that are coupled to a nucleus of high gyromagnetic ratio, usually 1H. The INEPT pulse sequence also forms the basis of many two-and three-dimensional heteronuclear NMR experiments. The optimal delay between transients in INEPT is determined by the T1 of the coupled proton. When the nucleus is coupled to a labile proton, however, the efficiency of the experiment is reduced if exchange with solvent protons becomes sufficiently rapid. This aspect can be used to measure the exchange rates of 15N-coupled protons in biomolecules, which, in turn, provides a useful source of structural and dynamic information. In the heteronucleusobserve proton-decoupled INEPT experiment, where all protons are irradiated during acquisition, the intensity of the INEPT signal is determined by the length of the relaxation delay between transients. When the exchange rate of the labile proton is of the order of 1/T1, the rate of recovery is reduced by transfer of saturation from the slowly recovering solvent protons, and the signal intensity at moderate relaxation delays is reduced. A second, faster-exchange regime is entered when the exchange rate of the coupled proton is of the order of J. The contribution of hydrogen exchange to the intensity of the decoupled INEPT signal has been determined by an extension of the theory of saturation transfer developed by Forsén and Hoffman (J. Chem. Phys.40, 1189-1196, 1963). The INEPT experiment has been used to measure the hydrogen exchange rate in a model amide, [15N]acetylglycine, and for several amides in site-selectively labeled M13 coat protein. © 1993 Academic Press, Inc.