An 1H-13C-13C-Edited 1H NMR Experiment for Making Resonance Assignments in the Active Site of Heme Proteins

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In paramagnetic heme proteins, it is often problematic to make proton resonance assignments for heme substituents that do not have large isotropic shifts and consequently lie under the large envelope of polypeptide resonances. Furthermore, assignments that would normally be performed with the aid of HMBC experiments in diamagnetic molecules can prove difficult in the active site of paramagnetic heme proteins if T-12 > 2JCH. To circumvent this problem, a new method is presented to selectively detect 1H in 1Hn-13C-13C fragments biosynthetically introduced into the active site of heme proteins. The pulse sequence combines well-known building blocks such as INEPT to transfer 1H spin magnetization to bonded 13C nuclei, followed by INADEQUATE to generate 13C-13C double-quantum coherence that is selected with pulsed field gradients, and finally reverse-INEPT to transfer magnetization back to 1H nuclei for subsequent observation. The new 1Hn-13C-13C edited experiment takes advantage of the relatively large values of 1JCH and 1JCC, avoiding the long interpulse delays in HMBC that compromise the detectability of rapidly relaxing nuclei. The potential applicability of the pulse sequence is demon strated by its contribution to the unambiguous assignment of the carbonyl carbons in the heme propionates of ferricytochrome b5. © 1998 Academic Press.

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Journal of Magnetic Resonance

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