The perfect carbon TOCSY (iPE TOCSY) facilitates assignment of large biomolecules
Abstract
Side chain assignments are essential for in-depth characterization of proteins. We present here two refined TOCSY experiments based on the use of a variant of “perfect echo” to partially suppress the negative effects arising from homonuclear 13C couplings in the indirect dimension: The iPE-(H)CCH-TOCSY for correlating aliphatic and aromatic side chains and the iPE-(H)C(CCO)NH-TOCSY for linking side chains, in particular methyl groups, to the backbone. The iPE-TOCSY pulse sequences result in spectra of higher sensitivity and higher resolution at the same time compared to widely used standard experiments. Higher sensitivity is achieved by delayed dephasing of the signal due to homonuclear couplings and via the COSY-type C-C transfer inherent in PE-schemes. For a protein like MBP (43 kDa), sensitivity increases of up to 3-fold are shown. For enhancing resolution, the iPE scheme allows prolonging the effectively usable t1 time from 11 to 16 ms, which leads to sharper lines and reduces overlap significantly. These two experiments have the potential to increase the size limit for side chain assignments and simplify spectral analysis by reducing overlap.
