A TROSY CPMG sequence for characterizing chemical exchange in large proteins.
A new NMR spin relaxation experiment is described for measuring chemical exchange time constants from approximately 0.5 ms to 5 ms in 15N-labeled macromolecules. The pulse sequence is based on the Carr-Purcell-Meiboom-Gill technique [Carr and Purcell (1954) Phys. Rev., 94, 630-638; Meiboom and Gill (1958) Rev. Sci. Instrum., 29, 688-691; Loria et al. (1999) J. Am. Chem. Soc., 121, 2331-2332], but implements TROSY selection [Pervushin et al. (1997) Proc. Natl. Acad. Sci. USA, 94, 12366-12371] to permit measurement of exchange linebroadening contributions to the narrower component of the 1H-15N scalar-coupled doublet. This modification extends the size limitation imposed on relaxation measurements due to the fast decay of transverse magnetization in larger macromolecules. The new TROSY-CPMG experiment is demonstrated on a [U-98% 15N] labeled sample of basic pancreatic trypsin inhibitor and a [U-83% 2H, U-98% 15N] labeled sample of triosephosphate isomerase, a 54 kDa homodimeric protein.
Loria
J P
JP
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Rance
M
M
Palmer
A G
AG
D001665:Binding Sites; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008280:Magnetics; D009038:Motion; D009587:Nitrogen Isotopes; D020799:Pancrelipase / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D011522:Protons; D014305:Triose-Phosphate Isomerase / Q000737:chemistry*; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D010940:Plant Proteins; D011522:Protons; D014361:Trypsin Inhibitors; D020799:Pancrelipase; D000516:alpha-Amylases; D014305:Triose-Phosphate Isomerase