An asymmetric deuterium labeling strategy to identify interprotomer and intraprotomer NOEs in oligomeric proteins.
A major difficulty in determining the structure of an oligomeric protein by NMR is the problem of distinguishing inter- from intraprotomer NOEs. In order to address this issue in studies of the 27 kD compact trimeric domain of the MHC class II-associated invariant chain, we compared the 13C NOESY-HSQC spectrum of a uniformly 13C-labeled trimer with the spectrum of the same trimer labeled with 13C in only one protomer, and with deuterium in the other two protomers. The spectrum of the unmixed trimer included both inter- and intraprotomer NOEs while the spectrum of the mixed trimer included only intraprotomer peaks. NOEs clearly absent from the spectrum of the mixed trimer could be confidently assigned to interprotomer interactions. Asymmetrically labeled trimers were isolated by refolding a 13C-labeled shorter form of the protein with a 2H-labeled longer form, chromatographically purifying trimers with only one short chain, and then processing the trypsin to yield only protomers with the desired N- and C-termini. In contrast to earlier studies, in which statistical mixtures of differently labeled protomers were analyzed, our procedure generated only a well-defined 1:2 oligomer, and no other mixed oligomers were present. This increased the maximum possible concentration of NMR-active protomers and thus the sensitivity of the experiments. Related methods should be applicable to many oligomeric proteins, particularly those with slow protomer exchange rates.
Jasanoff
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Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry / Q000235:genetics; D003903:Deuterium; D004926:Escherichia coli / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; C045084:invariant chain; D003903:Deuterium