13C−13C Rotational Resonance Width Distance Measurements in Uniformly 13C-Labeled Peptides

Abstract

The rotational resonance width (R²W) experiment is a constant-time version of the rotational resonance (R²) experiment, in which the magnetization exchange is measured as a function of sample spinning frequency rather than the mixing time. The significant advantage of this experiment over conventional R² is that both the dipolar coupling and the relaxation parameters can be independently and unambiguously extracted from the magnetization exchange profile. In this paper, we combine R²W with two-dimensional ¹³C−¹³C chemical shift correlation spectroscopy and demonstrate the utility of this technique for the site-specific measurement of multiple ¹³C−¹³C distances in uniformly labeled solids. The dipolar truncation effects, usually associated with distance measurements in uniformly labeled solids, are considerably attenuated in R²W experiments. Thus, R²W experiments are applicable to uniformly labeled biological systems. To validate this statement, multiple ¹³C−¹³C distances (in the range of 3−6 Å) were determined in N-acetyl-[U-¹³C,¹⁵N]l-Val-l-Leu with an average precision of ±0.5 Å. Furthermore, the distance constraints extracted using a two-spin model agree well with the X-ray crystallographic data.