Measurement of Dipolar Cross-Correlation in Methylene Groups in Uniformly 13 C-, 15 N-Labeled Proteins

A CC(CO)NH TOCSY-based 3D pulse scheme is presented for measuring 1 H- 13 C dipole-dipole cross-correlated relaxation at CH 2 positions in uniformly 13 C-, 15 N-labeled proteins. Simulations based on magnetization evolution under relaxation and scalar coupling interactions show that cross-correlation rates between 1 H- 13 C dipoles in CH 2 groups can be simply obtained from the intensities of 13 C triplets. The normalized cross-correlation relaxation rates are related to cross-correlation order parameters for macromolecules undergoing isotropic motion, which reflect the degrees of spatial restriction of CH 2 groups. The study on human intestinal fatty acid binding protein (131 residues) in the presence of oleic acid demonstrates that side chain dynamics at most CH 2 positions can be characterized for proteins less than 15 kDa in size, with the proposed TOCSY-based approach.