Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: application to a 723-residue enzyme.
A new CPMG-based multiple quantum relaxation dispersion experiment is presented for measuring millisecond dynamic processes at side-chain methyl positions in high molecular weight proteins. The experiment benefits from a methyl-TROSY effect in which cancellation of intramethyl dipole fields occurs, leading to methyl (13)C-(1)H correlation spectra of high sensitivity and resolution (Tugarinov, V.; Hwang, P. M.; Ollerenshaw, J. E.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 10420-10428). The utility of the methodology is illustrated with an application to a highly deuterated, methyl-protonated sample of malate synthase G, an 82 kDa enzyme consisting of a single polypeptide chain. A comparison of the sensitivity obtained using the present approach relative to existing HSQC-type (13)C single quantum dispersion experiments shows a gain of a factor of 5.4 on average, significantly increasing the range of applications for this methodology.
Korzhnev
Dmitry M
DM
Protein Engineering Network Centres of Excellence and the Departments of Medical Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Kloiber
Karin
K
Kanelis
Voula
V
Tugarinov
Vitali
V
Kay
Lewis E
LE
D002247:Carbon Isotopes; D008292:Malate Synthase / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D013816:Thermodynamics
D002247:Carbon Isotopes; D008292:Malate Synthase