Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data.

Abstract

To test whether distances derived from paramagnetic broadening of (15)N heteronuclear single quantum coherence (HSQC) resonances could be used to determine the global fold of a large, perdeuterated protein, we used site-directed spin-labeling of 5 amino acids on the surface of (15)N-labeled eukaryotic translation initiation factor 4E (eIF4E). eIF4E is a 25 kDa translation initiation protein, whose solution structure was previously solved in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) micelle of total molecular mass approximately 45-50 kDa. Distance-dependent line broadening consistent with the three-dimensional structure of eIF4E was observed for all spin-label substitutions. The paramagnetic broadening effects (PBEs) were converted into distances for modeling by a simple method comparing peak heights in (15)N-HSQC spectra before and after reduction of the nitroxide spin label with ascorbic acid. The PBEs, in combination with HN-HN nuclear Overhauser effects (NOEs) and chemical shift index (CSI) angle restraints, correctly determined the global fold of eIF4E with a backbone precision of 2.3 A (1.7 A for secondary structure elements). The global fold was not correctly determined with the HN-HN NOEs and CSI angles alone. The combination of PBEs with simulated restraints from another nuclear magnetic resonance (NMR) method for global fold determination of large proteins (methyl-protonated, highly deuterated samples) improved the quality of calculated structures. In addition, the combination of the two methods simulated from a crystal structure of an all alpha-helical protein (40 kDa farnesyl diphoshphate synthase) correctly determined the global fold where neither method individually was successful. These results show the potential feasibility of obtaining medium-resolution structures for proteins in the 40-100 kDa range via NMR.