SEQUENTIAL RESONANCE ASSIGNMENT OF MEDIUM-SIZED N-15 C-13-LABELED PROTEINS WITH PROJECTED 4D TRIPLE-RESONANCE NMR EXPERIMENTS/

We recently introduced a new line of reduced-dimensionality experiment s making constructive use of axial peak magnetization, which has so fa r been suppressed as an undesirable artifact in multidimensional NMR s pectra [Szyperski, T., Braun, D., Banecki, B. and Wuthrich, K. (1996) J. Am. Chem. Sec., 118, 8146-8147]. The peaks arising from the axial m agnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensiona l (4D) triple resonance experiments for the efficient sequential reson ance assignment of N-15/C-13-labeled proteins. A 3D (H) under bar(alph a/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 combinat ion yields sequential assignments based on the measurement of C-13(alp ha) chemical shifts and provides a complete H-1, C-13 and N-15 resonan ce assignment of polypeptide backbone and CHnbeta moieties, When emplo ying 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 fas hion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile m utual interconversion of single-quantum and zero/double-quantum freque ncies detected in conventional and reduced-dimensionality spectra, res pectively. In particular, SPSCAN comprises a peak picking routine tail ored to cope with the distinct peak patterns of projected NMR experime nts performed with simultaneous acquisition of central peaks. Data wer e acquired at 13 degrees C for the N-terminal 63-residue polypeptide f ragment of the 434 repressor. Analysis of these spectra, which are rep resentative 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 p roteins.