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APSY-NMR with proteins: practical aspects and backbone assignment

Academic Article
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Overview

authors

  • Hiller, S.
  • Wider, G.
  • Wuthrich, Kurt

publication date

  • November 2008

journal

  • Journal of Biomolecular NMR  Journal

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.

subject areas

  • Algorithms
  • Amino Acid Sequence
  • Amino Acids
  • Molecular Weight
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptides
  • Proteins
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrum Analysis
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Research

keywords

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

International Standard Serial Number (ISSN)

  • 0925-2738

Digital Object Identifier (DOI)

  • 10.1007/s10858-008-9266-y

PubMed ID

  • 18841481
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Additional Document Info

start page

  • 179

end page

  • 195

volume

  • 42

issue

  • 3

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