Selective `unlabeling' of amino acids in fractionally 13C labeled proteins: An approach for stereospecific NMR assignments of CH3 groups in Val and Leu residues |
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Authors: | H.S. Atreya K.V.R. Chary |
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Affiliation: | (1) Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai, 400005, India |
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Abstract: | A novel methodology for stereospecific NMR assignments of methyl (CH3) groups of Val and Leu residues in fractionally 13C-labeled proteins is presented. The approach is based on selective `unlabeling' of specific amino acids in proteins while fractionally 13C-labeling the rest. A 2D [13C-1H] HSQC spectrum recorded on such a sample is devoid of peaks belonging to the `unlabeled' amino acid residues. Such spectral simplification aids in unambiguous stereospecific assignment of diastereotopic CH3 groups in Val and Leu residues in large proteins. This methodology has been demonstrated on a 15 kDa calcium binding protein from Entamoeba histolytica (Eh-CaBP). |
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Keywords: | amino acid biosynthesis amino acid unlabeling coupling constant diastereotopic methyl groups HSQC isotope labeling multidimensional NMR stereospecific assignments |
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