title: 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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Battiste
authors.forename: G
authors.initials: G
authors.lastname: Wagner
mesh_terms: D000595:Amino Acid Sequence; D002793:Cholic Acids / Q000737:chemistry; D003903:Deuterium; D039561:Eukaryotic Initiation Factor-4E; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010448:Peptide Initiation Factors / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D013113:Spin Labels; D015003:Yeasts / Q000737:chemistry
chemical_list: D002793:Cholic Acids; D039561:Eukaryotic Initiation Factor-4E; D009587:Nitrogen Isotopes; D010448:Peptide Initiation Factors; D011506:Proteins; D013113:Spin Labels; D003903:Deuterium; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate