Journal of Biomolecular NMR

, Volume 42, Issue 3, pp 179–195 | Cite as

APSY-NMR with proteins: practical aspects and backbone assignment

Article

Abstract

Automated projection spectroscopy (APSY) is an NMR technique for the recording of discrete sets of projection spectra from higher-dimensional NMR experiments, with automatic identification of the multidimensional chemical shift correlations by the dedicated algorithm GAPRO. This paper presents technical details for optimizing the set-up and the analysis of APSY-NMR experiments with proteins. Since experience so far indicates that the sensitivity for signal detection may become the principal limiting factor for applications with larger proteins or more dilute samples, we performed an APSY-NMR experiment at the limit of sensitivity, and then investigated the effects of varying selected experimental parameters. To obtain the desired reference data, a 4D APSY-HNCOCA experiment with a 12-kDa protein was recorded in 13 min. Based on the analysis of this data set and on general considerations, expressions for the sensitivity of APSY-NMR experiments have been generated to guide the selection of the projection angles, the calculation of the sweep widths, and the choice of other acquisition and processing parameters. In addition, a new peak picking routine and a new validation tool for the final result of the GAPRO spectral analysis are introduced. In continuation of previous reports on the use of APSY-NMR for sequence-specific resonance assignment of proteins, we present the results of a systematic search for suitable combinations of a minimal number of four- and five-dimensional APSY-NMR experiments that can provide the input for algorithms that generate automated protein backbone assignments.

Keywords

Projection spectroscopy Automated peak picking Protein NMR Sequence-specific NMR assignments 

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Sebastian Hiller
    • 1
    • 2
  • Gerhard Wider
    • 1
  • Kurt Wüthrich
    • 1
    • 3
  1. 1.Institute of Molecular Biology and BiophysicsETH ZürichZürichSwitzerland
  2. 2.Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUSA
  3. 3.Department of Molecular Biology and Skaggs Institute of Chemical BiologyThe Scripps Research InstituteLa JollaUSA

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