Chain-selective isotopic labeling for NMR studies of large multimeric proteins: Application to hemoglobin

Multidimensional, multinuclear NMR has the potential to elucidate the mecha nisms of allostery and cooperativity in multimeric proteins under near-phys iological conditions. However, NMR studies of proteins made up of non-equiv alent subunits face the problem of severe resonance overlap, which can prev ent the unambiguous assignment of resonances, a necessary step in interpret ing the spectra. We report the application of a chain-selective labeling te chnique, in which one type of subunit is labeled at a time, to carbonmonoxy -hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to t he physiological function of hemoglobin. Among these amino acid residues ar e the surface histidyls, which account for the majority of the Bohr effect. in the present work, we report the results of two-dimensional heteronuclea r multiple quantum coherence (HMQC) experiments performed on recombinant N- 15-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shi fts, these spectra also reveal the corresponding C4-proton (H delta(2)) res onances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also all ows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resona nces of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopi c labeling, can provide resonance assignments of key regions in large, mult imeric proteins, suggesting an approach to elucidating the solution structu re of hemoglobin, a protein with molecular weight 64.5 kDa.