Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in (15)N-(13)C-ILV methyl labeled proteins.
We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record (13)C- and (15)N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field where even longer experimental times would be required for comparable resolution in NOESY-HSQC experiments. The method is demonstrated at 900 MHz and at 750 MHz on 37 and 31 kDa proteins, respectively. The resolution and time saving provided in this experiment was crucial for solving the structures of these two proteins.
Frueh
Dominique P
DP
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Leed
Alison
A
Arthanari
Haribabu
H
Koglin
Alexander
A
Walsh
Christopher T
CT
Wagner
Gerhard
G
D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010453:Peptide Synthases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
D011506:Proteins; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase