α&β HSQC, an HSQC-Type Experiment with Improved Resolution for I2S Groups

A new HSQC-type pulse scheme is introduced that effectively improves the spectral resolution by what corresponds to the size of geminalJHHcoupling constants, i.e., on the order of −14 Hz in CH2groups of peptides and proteins. The gain in resolution comes from the fact that, in I2S groups with I-spin detection, only those resonances with the passive I spin being in either the α or the β state are excited. The experiment α HSQC has two versions: one that is α and β selective in the echo and antiecho parts, respectively, or the opposite combination. This selection of polarizing only one half of the I-spin resonances works even when the two I spins are strongly coupled. Pure-absorption, selective α HSQC or β HSQC spectra are obtained by the two combinations of the echo part of one of the α HSQC spectra with the antiecho part of the other. These two spectra (α HSQC and β HSQC) can be merged into another spectrum, Mab HSQC, which has the appearance of an HSQC spectrum with homonuclear broadband decoupling of geminal scalar interactions. The method is demonstrated on aqueous solutions of a decapeptide at the natural-abundance level of isotopes and on the protein rhodniin (103 residues) uniformly labeled with13C and15N.