Stereospecific assignment of the NH2 resonances from the primary amides of asparagine and glutamine side chains in isotopically labeled proteins.
An HMQC-based pulse scheme is presented for the stereospecific assignment of asparagineand glutamine side-chain amide protons. The approach makes use of the recently developedquantitative-J correlation spectroscopy [Bax, A. et al. (1994) Methods Enzymol., 239,79-105] to distinguish the E and Z primary amide protons and, as such, eliminates theneed for assignments derived from more time-consuming and potentially ambiguous NOEmethods. An application of this method to a uniformly 15N,13C-labeled cellulose-bindingdomain is presented. When used in combination with a NOESY-HSQC experiment, thepredominant chi2 dihedral angles of two asparagine side chains in this protein can also bedefined.
McIntosh
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LP
Protein Engineering Network Centers of Excellence, Department of Biochemistry and Molecular Biology and Department of Chemistry, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3.
Brun
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E
Kay
L E
LE