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 Nz into 2NzHz and 2NzHz into -Nz 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 Nz and 2NzHz 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 15Nz-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.
Sahu
Debashish
D
Department of Biochemistry and Molecular Biology and Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0647, USA.
Clore
G Marius
GM
Iwahara
Junji
J
D003198:Computer Simulation; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
D018398:Homeodomain Proteins; D013912:Threonine; D004247:DNA