title: Automatic assignment of protein backbone resonances by direct spectrum inspection in targeted acquisition of NMR data.
abstract: The necessity to acquire large multidimensional datasets, a basis for assignment of NMR resonances, results in long data acquisition times during which substantial degradation of a protein sample might occur. Here we propose a method applicable for such a protein for automatic assignment of backbone resonances by direct inspection of multidimensional NMR spectra. In order to establish an optimal balance between completeness of resonance assignment and losses of cross-peaks due to dynamic processes/degradation of protein, assignment of backbone resonances is set as a stirring criterion for dynamically controlled targeted nonlinear NMR data acquisition. The result is demonstrated with the 12 kDa (13)C,(15) N-labeled apo-form of heme chaperone protein CcmE, where hydrolytic cleavage of 29 C-terminal amino acids is detected. For this protein, 90 and 98% of manually assignable resonances are automatically assigned within 10 and 40 h of nonlinear sampling of five 3D NMR spectra, respectively, instead of 600 h needed to complete the full time domain grid. In addition, resonances stemming from degradation products are identified. This study indicates that automatic resonance assignment might serve as a guiding criterion for optimal run-time allocation of NMR resources in applications to proteins prone to degradation.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore.
authors.forename: Leo E
authors.initials: LE
authors.lastname: Wong
authors.forename: James E
authors.initials: JE
authors.lastname: Masse
authors.forename: Victor
authors.initials: V
authors.lastname: Jaravine
authors.forename: Vladislav
authors.initials: V
authors.lastname: Orekhov
authors.forename: Konstantin
authors.initials: K
authors.lastname: Pervushin
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation
chemical_list: D001425:Bacterial Outer Membrane Proteins; C115384:CcmE protein, E coli; D029968:Escherichia coli Proteins; D006420:Hemeproteins
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