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 Wuthrich, K. (1996) J. Am. Chem. Sec., 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 N-15/C-13-labeled proteins. A 3D (H) under bar(alpha/beta) (C) under bar(alpha/beta)(CO)NHN experiment is recorded either in conjunction with 3D HNN <<(CO)under bar>,<(CA)under bar>> or with the newly presented 3D HNN<(CAHA)under bar> scheme. The first combination yields sequential assignments based on the measurement of C-13(alpha) chemical shifts and provides a complete H-1, C-13 and N-15 resonance assignment of polypeptide backbone and CHnbeta moieties, When employing the second combination, C-13=O chemical shifts are not measured, but the sequential assignment relies on both C-13(alpha) and H-1(alpha) 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 degrees 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 degrees C, demonstrates the efficiency of our approach for the assignment of medium-sized N-15/C-13 doubly labeled proteins.