A Combinatorial Selective Labeling Method for the Assignment of Backbone Amide NMR Resonances

• Published in: Journal of the American Chemical Society Vol. 126; no. 16; pp. 5020 - 5021
• Main Authors: Parker, Martin J, Aulton-Jones, Marc, Hounslow, Andrea M, Craven, C. Jeremy
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 28-04-2004
• Subjects: Amides - chemistry; Amino Acids - chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbon Isotopes; Deuterium; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Green Fluorescent Proteins; Isotope Labeling - methods; Ligands; Luminescent Proteins - chemistry; Magnetic Resonance Spectroscopy - methods; Nitrogen Isotopes; Proteins; Biochemical analysis; Nitrogen 15; Green fluorescent protein; NMR spectrometry; Carbon 13; Heteronuclear correlation; Structural analysis; Two dimension spectrometry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja039601r

A combinatorial selective labeling (CSL) method is presented for the assignment of backbone amide NMR resonances, which has a particular application in the identification of protein−ligand interaction sites. The method builds on the dual amino acid selective labeling technique. In the CSL method a number of different samples are produced, each with a different pattern of labeled amino acids. By analyzing peak intensities in HSQC and 2D HNCO spectra of these samples, a large number of combinations of amino acid pairs can be simultaneously assigned. We demonstrate the method on the 27 kDa protein GFP. The samples can be produced rapidly and cost-effectively in a commercially available in vitro translation system. The method greatly simplifies the process of backbone assignment and would be very straightforward to automate.