An exchange-free measure of 15N transverse relaxation: An NMR spectroscopy application to the study of a folding intermediate with pervasive chemical exchange

Abstract

A series of experiments are presented that provide an exchange-free measure of dipole-dipole 15N transverse relaxation, Rdd, that can then be substituted for 15N R1ρ or R2 rates in the study of internal protein dynamics. The method is predicated on the measurement of a series of relaxation rates involving 1H- 15N longitudinal order, anti-phase 1H and 15N single-quantum coherences, and 1H-15N 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 Rdd 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 b562 that shows large contributions to 15N R1ρ relaxation from chemical exchange. © 2007 American Chemical Society.