An exchange-free measure of 15N transverse relaxation: an NMR spectroscopy application to the study of a folding intermediate with pervasive chemical exchange.
A series of experiments are presented that provide an exchange-free measure of dipole-dipole (15)N transverse relaxation, R(dd), that can then be substituted for (15)N R(1rho) or R(2) rates in the study of internal protein dynamics. The method is predicated on the measurement of a series of relaxation rates involving (1)H-(15)N longitudinal order, anti-phase (1)H and (15)N single-quantum coherences, and (1)H-(15)N multiple quantum coherences; the relaxation rates of all coherences are measured under conditions of spin-locking. Results from detailed simulations and experiments on a number of protein systems establish that R(dd) values are independent of exchange and systematic errors from dipolar interactions with proximal protons are calculated to be less than 1-2%, on average, for applications to perdeuterated proteins. Simulations further indicate that the methodology is rather insensitive to the exact level of deuteration so long as proteins are reasonably highly deuterated (>50%). The utility of the methodology is demonstrated with applications involving protein L, ubiquitin, and a stabilized folding intermediate of apocytochrome b(562) that shows large contributions to (15)N R(1rho) relaxation from chemical exchange.
Hansen
D Flemming
DF
Department of Medical Genetics, The University of Toronto, Toronto, Ontario, Canada, M5S 1A8.
Yang
Daiwen
D
Feng
Haniqiao
H
Zhou
Zheng
Z
Wiesner
Silke
S
Bai
Yawen
Y
Kay
Lewis E
LE
D003198:Computer Simulation; D008956:Models, Chemical; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D017510:Protein Folding; D025801:Ubiquitin / Q000737:chemistry
D009587:Nitrogen Isotopes; D025801:Ubiquitin