A TROSY CPMG sequence for characterizing chemical exchange in large proteins

A new NMR spin relaxation experiment is described for measuring chemical ex change time constants from approximately 0.5 ms to 5 ms in N-15-labeled mac romolecules. The pulse sequence is based on the Carr-Purcell-Meiboom-Gill t echnique [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. S oc., 121, 2331-2332], but implements TROSY selection [Pervushin et al. (199 7) Proc. Natl. Acad. Sci. USA, 94, 12366-12371] to permit measurement of ex change linebroadening contributions to the narrower component of the H-1-N- 15 scalar-coupled doublet. This modification extends the size limitation im posed on relaxation measurements due to the fast decay of transverse magnet ization in larger macromolecules. The new TROSY-CPMG experiment is demonstr ated on a [U-98% N-15] labeled sample of basic pancreatic trypsin inhibitor and a [U-83% H-2, U-98% N-15] labeled sample of triosephosphate isomerase, a 54 kDa homodimeric protein.