Probing slow backbone dynamics in proteins using TROSY-based experiments to detect cross-correlated time-modulation of isotropic chemical shifts

The difference in the relaxation rates of zero-quantum (ZQ) and double-quantum (DQ) coherences is the result of three principal mechanisms. These include the cross-correlation between the chemical shift anisotropies of the two participating nuclei, dipolar interactions with remote protons as well as interference effects due to the time-modulation of their isotropic chemical shifts as a consequence of slow mus-ms dynamics. The last effect when present, dominates the others resulting in large differences between the relaxation rates of ZQ and DQ coherences. We present here four sets of TROSY-based (Salzmann et al., 1998) experiments that measure this effect for several pairs of backbone nuclei including N-15, C-13(alpha) and C-13'. These experiments allow the detection of the presence of slow dynamic processes in the protein backbone including correlated motion over two and three bonds. Further, we define a new parameter chi which represents the extent of correlated motion on the slow (mus-ms) timescale. This methodology has been applied to N-15, C-13, REDPRO-H-2-labeled (Shekhtman et al., 2002) human ubiquitin. The ubiquitin backbone is seen to exhibit extensive dynamics on the slow timescale. This is most pronounced in several residues in the N-terminal region of the alpha- helix and in the loop connecting the strands beta(4) and beta(5). These residues which include Glu24, Asn25, Glu51 and Asp 52 form a continuous surface. As an additional benefit, the measured rates confirm the dependence of the C-13(alpha) chemical shift tensor on local secondary structure of the protein backbone.