A combinatorial selective labeling method for the assignment of backbone amide NMR resonances.
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.
Parker
Martin J
MJ
Department of Molecular Biology and Biotechnology, University of Sheffield, U.K. m.j.parker@leeds.ac.uk
Aulton-Jones
Marc
M
Hounslow
Andrea M
AM
Craven
C Jeremy
CJ
D000577:Amides / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry; D002247:Carbon Isotopes; D003903:Deuterium; D049452:Green Fluorescent Proteins; D007553:Isotope Labeling / Q000379:methods; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes
D000577:Amides; D000596:Amino Acids; D002247:Carbon Isotopes; D008024:Ligands; D008164:Luminescent Proteins; D009587:Nitrogen Isotopes; D049452:Green Fluorescent Proteins; D003903:Deuterium