A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes.
A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described. (13)C/(1)H-I,L,V-methyl, U-(2)H labeled protein is produced using the biosynthetic precursors [gamma-(13)C]-alpha-ketobutyrate and [gamma,gamma'-(13)C(2)]-alpha-ketoisovalerate. The labeled protein is mixed with its unlabeled binding partner and a 3D (13)C-HMQC-NOESY is recorded, yielding unambiguous intermolecular aromatic/methyl NOEs. A simple synthesis of the biosynthetic precursors via reaction of diethyl oxalate with alkyl Grignard compounds is reported. The method is demonstrated for a 35 kDa heterodimeric protein complex dissolved in a CHAPS micelle. This approach will facilitate the solution structure determination of protein/protein, protein/ligand or protein/nucleic acid complexes.
Gross
John D
JD
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave. Boston, MA 02115, USA.
Gelev
Vladimir M
VM
Wagner
Gerhard
G
D002087:Butyrates / Q000737:chemistry; D002793:Cholic Acids / Q000737:chemistry; D039561:Eukaryotic Initiation Factor-4E / Q000032:analysis; D045782:Hemiterpenes; D007554:Isotopes; D007651:Keto Acids / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008823:Micelles; D008956:Models, Chemical; D010070:Oxalates / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012680:Sensitivity and Specificity; D013223:Statistics as Topic
D002087:Butyrates; D002793:Cholic Acids; D039561:Eukaryotic Initiation Factor-4E; D045782:Hemiterpenes; D007554:Isotopes; D007651:Keto Acids; D008024:Ligands; D008823:Micelles; D010070:Oxalates; D011506:Proteins; C005087:alpha-ketobutyric acid; C001505:alpha-ketoisovalerate; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate