DETERMINATION OF LOCAL LIGAND CONFORMATIONS IN SLOWLY TUMBLING PROTEINS BY HOMONUCLEAR 2D AND 3D NMR - APPLICATION TO HEME PROPIONATES IN LEGHEMOGLOBIN

A novel procedure for proton assignment and determination of the confo rmation of the heme propionates of heme proteins is described. Analysi s of 3D TOCSY-NOESY and NOESY-NOESY NMR spectra of carbonmonoxy leghem oglobin (LbCO) allowed the unambiguous identification of the spin syst ems of both heme propionates. Long-range ROE-type transfers observed i n 3D TOCSY-NOESY and 2D TOCSY spectra have been found to be useful for identifying the heme propionate protons. NOE buildup curves have been measured and provide evidence for spin diffusion at mixing times less than 50 ms. Cross-peak volumes and J coupling information extracted f rom a 30-ms 2D NOESY and a 2QF-COSY spectrum, respectively, have been used for the determination of the propionate assignments and conformat ions. For both propionates, an independent grid search about two rotat able torsion angles performed with a 10-degrees increment allowed samp ling of 1296 different conformations. Intra-heme NOE cross-relaxation rates and 3J coupling constants have been back-calculated for each of these conformations by taking into account all possible assignments, i ncluding stereospecific assignments, and compared with the experimenta l data. Additional NOEs to the globin allow discrimination between pro pionate mirror images with respect to the heme plane. Conformations we re obtained that are a good fit to the experimental data, providing un ambiguous (stereo) assignments for the propionate methylene proton res onances. The propionate conformations determined by NMR are compared w ith those found in crystal structures of lupin leg hemoglobin (Lb) and other heme proteins. Possible hydrogen-bonding interactions involving the propionate carboxyl groups and globin side chains are discussed. This study demonstrates the possibility of detailed local structural c haracterization of a ligand embedded in a slowly tumbling protein by h omonuclear NMR using only limited qualitative distance information to the protein.