A systematic analysis of backbone amide assignments achieved via combinatorial selective labelling of amino acids.
With the advent of high-yield cell-free expressions systems, many researchers are exploiting selective isotope labelling of amino acids to increase the efficiency and accuracy of the NMR assignment process. We developed recently a combinatorial selective labelling (CSL) method capable of yielding large numbers of residue-type and sequence-specific backbone amide assignments, which involves comparing cross-peak intensities in 1H- 15N HSQC and 2D 1H- 15N HNCO spectra collected for five samples containing different combinations of 13C- and 15N-labelled amino acids [Parker MJ, Aulton-Jones M, Hounslow A, Craven C J (2004) J Am Chem Soc 126:5020-5021]. In this paper we develop a robust method for establishing the reliability of these assignments. We have performed a detailed statistical analysis of the CSL data collected for a model system (the B1 domain of protein G from Streptococcus), developing a scoring method which allows the confidence in assignments to be assessed, and which enables the effects of overlap on assignment fidelity to be predicted. To further test the scoring method and also to assess the performance of CSL in relation to sample quality, we have applied the method to the CSL data collected for GFP in our previous study.
Jeremy Craven
C
C
Department of Biotechnology and Molecular Biology, University of Sheffield, Sheffield, S10 2TN, UK.
Al-Owais
Moza
M
Parker
Martin J
MJ
D000577:Amides / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular; D012107:Research Design; D013291:Streptococcus / Q000737:chemistry*
D000577:Amides; D000596:Amino Acids; D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins