Sequential resonance assignment of medium-sized ¹⁵N/¹³C-labeled proteins with projected 4D triple resonance NMR experiments

We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146-8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of ¹⁵N/¹³C-labeled proteins. A 3D Hα/β Cα/β(CO)NHN experiment is recorded either in conjunction with 3D HNN〈CO,CA〉 or with the newly presented 3D HNNCAHA scheme. The first combination yields sequential assignments based on the measurement of ¹³C^α chemical shifts and provides a complete ¹H, ¹³C and ¹⁵N resonance assignment of polypeptide backbone and CH^β_n moieties. When employing the second combination, ¹³C=O chemical shifts are not measured, but the sequential assignment relies on both ¹³Cα and ¹H^α chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13°C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30°C, demonstrates the efficiency of our approach for the assignment of medium-sized ¹⁵N/¹³C doubly labeled proteins.