ISSN:
1573-5001
Keywords:
automated resonance assignment; NMR assignments; reduced-dimensionality triple-resonance experiments; resolution enhancement in NMR.
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract 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 $$\underline {\rm{H}}$$ α/β $$\underline {\rm{C}}$$ α/β(CO)NHN experiment is recorded either in conjunction with 3D HNN〈 $$\underline {{\rm{CO}}} {\rm{,}}\underline {{\rm{CA}}}$$ 〉 or with the newly presented 3D HNN $${\rm{CAHA}}$$ scheme. The first combination yields sequential assignments based on the measurement of13 Cα chemical shifts and provides a complete 1H, 13C and 15N resonance assignment of polypeptide backbone and CHn β moieties. When employing the second combination, 13C=O chemical shifts are not measured, but the sequential assignment relies on both 13Cα and1 Hα 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-sized15 N/13C doubly labeled proteins.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1008287921055
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