An advantage for use of isotope labeling and NMR chemical shifts to analyze the structure of four homologous IgG-binding domains of staphylococcal protein A

Because of the complexity arising from the large molecular size and the ami no acid sequence homologies of IgG-binding domains of Staphylococcal Protei n A (SpA), we have introduced a combination of stable isotope labeling and both qualitative and quantitative investigations of the structural dependen ce of the NMR chemical shifts for its structure analysis. In order to enabl e selective isotope labeling with high efficiency, a mutated low molecular weight Protein A (LPA; MWt = 27 kDa) which consists of E, D, A, B and 13 re sidues of the C-domain was used in this study. Amide proton chemical shifts , measured using uniformly N-15-labeled LPA and LPA labeled selectively wit h N-15-alanine, show that the turn between helices 1 and 2, and its tertiar y interactions with helix 3, are very similar in all domains. This contradi cts previous results obtained using independent structure calculations on i solated domains. The close similarity in NH and N-15 chemical shifts of ala nine residues in the interdomain linker suggests that the linker maintains a similar structure both in isolated domains and in the intact protein. We show that the high-field shifted methyl signal of Ala 48 is affected by the ring-current effect arising from Phe 30, and has a very similar helical en vironment in all four domains. Thus, helix 3 is present in all domains, as we previously reported [Kikuchi et al., J Biochem Biophys Method, 1999:38:2 03-208], even though it is not observed in the crystal structure [Deisenhof er J. Biochemistry 1981;20:2361-2370]. (C) 2000 Elsevier Science B.V. All r ights reserved.