TROSY-based z-exchange spectroscopy: Application to the determination of the activation energy for intermolecular protein translocation between specific sites on different DNA molecules

A two-dimensional TROSY-based z-exchange ¹H-¹⁵N correlation experiment for the quantitative analysis of kinetic processes in the slow exchange regime is presented. The pulse scheme converts the product operator terms N_z into 2N_zH_z and 2N _zH_z into -N_Z in the middle of the z-mixing period, thereby suppressing the buildup of spurious semi-TROSY peaks arising from the different relaxation rates for the N_z and 2N _zH_z terms and simplifying the behavior of longitudinal magnetization for an exchanging system during the mixing period. Theoretical considerations and experimental data demonstrate that the TROSY-based z-exchange experiment permits quantitative determination of rate constants using the same procedure as that for the conventional non-TROSY ¹⁵N _z-exchange experiment. Line narrowing as a consequence of the use of the TROSY principle makes the method particularly suitable for kinetic studies at low temperature, thereby permitting activation energies to be extracted from data acquired over a wider temperature range. We applied this method to the investigation of the process whereby the HoxD9 homeodomain translocates between specific target sites on different DNA molecules via a direct transfer mechanism without going through the intermediary of free protein. The activation enthalpy for intermolecular translocation was determined to be 17 kcal/mol.