TROSY-Based z-Exchange Spectroscopy:  Application to the Determination of the Activation Energy for Intermolecular Protein Translocation between Specific Sites on Different DNA Molecules

• Published in: Journal of the American Chemical Society Vol. 129; no. 43; pp. 13232 - 13237
• Main Authors: Sahu, Debashish, Clore, G. Marius, Iwahara, Junji
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 31.10.2007
• Subjects: Computer Simulation; DNA - chemistry; DNA - metabolism; Homeodomain Proteins - chemistry; Homeodomain Proteins - metabolism; Magnetic Resonance Spectroscopy - methods; Models, Molecular; Nucleic Acid Conformation; Protein Structure, Tertiary; Threonine - chemistry; Threonine - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja074604f

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 z H z and 2N z H 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 z H 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.