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

To test whether distances derived from paramagnetic broadening of N-15 hete ronuclear single quantum coherence (HSQC) resonances could be used to deter mine the global fold of a large, perdeuterated protein, we used site-direct ed spin-labeling of 5 amino acids on the surface of N-15-labeled eukaryotic translation initiation factor 4E (eIF4E). eIF4E is a 25 kDa translation in itiation protein, whose solution structure was previously solved in a 3-[(3 -cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate (CHAPS) micel le of total molecular mass similar to 45-50 kDa. Distance-dependent Line br oadening consistent with the three-dimensional structure of eIF4E was obser ved for all spin-label substitutions. The paramagnetic broadening effects ( PBEs) were converted into distances for modeling by a simple method compari ng peak heights in N-15-HSQC spectra before and after reduction of the nitr oxide spin label with ascorbic acid. The PBEs, in combination with HN-HN nu clear Overhauser effects (NOEs) and chemical shift index (CSI) angle restra ints, correctly determined the global fold of eIF4E with a backbone precisi on of 2.3 Angstrom (1.7 Angstrom for secondary structure elements). The glo bal fold was not correctly determined with the HN-HN NOEs and CSI angles al one. The combination of PBEs with simulated restraints from another nuclear magnetic resonance (NMR) method for global fold determination of large pro teins (methyl-protonated, highly deuterated samples) improved the quality o f calculated structures. In addition, the combination of the two methods si mulated from a crystal structure of an all alpha-helical protein (40 kDa fa rnesyl diphoshphate synthase) correctly determined the global fold where ne ither method individually was successful. These results show the potential feasibility of obtaining medium-resolution structures for proteins in the 4 0-100 kDa range via NMR.