Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection.
A pair of HN-methyl NOESY experiments that are based on simultaneous TROSY-type detection of amide and methyl groups is described. The preservation of cross-peak symmetry in the simultaneous (1)H-(15)N/(13)CH(3) NOE spectra enables straightforward assignments of HN-methyl NOE cross-peaks in large and complex protein structures. The pulse schemes are designed to preserve the slowly decaying components of both (1)H-(15)N and methyl (13)CH(3) spin-systems in the course of indirect evolution (t (2)) and acquisition period (t (3)) of 3D NOESY experiments. The methodology has been tested on {U-[(15)N,(2)H]; Iledelta1-[(13)CH(3)]; Leu,Val-[(13)CH(3),(12)CD(3)]}-labeled 82-kDa enzyme Malate Synthase G (MSG). A straightforward procedure that utilizes the symmetry of NOE cross-peaks in the time-shared 3D NOE data sets allows unambiguous assignments of more than 300 HN-methyl interactions in MSG from a single 3D data set providing important structural restraints for derivation of the backbone global fold.
Guo
Chenyun
C
Department of Chemistry and Biochemistry, University of Maryland, College Park, 20742, USA.
Tugarinov
Vitali
V
D000465:Algorithms; D000577:Amides / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D008292:Malate Synthase / Q000737:chemistry; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
D000577:Amides; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D008292:Malate Synthase