Theoretical analyses of the transferred cross-saturation method

Large molecules, such as membrane proteins, play crucial roles in various biologically important events. We have developed the transferred cross-saturation (TCS) method, which enables the identification of the contact residues of protein ligands in large complexes. However, rational optimization of the experimental conditions for the TCS method has been hampered by the lack of information about the influence of each experimental parameter on the observed TCS effects. Here, we established the theoretical description of the TCS method, which explicitly incorporated the isotopomers in the sample solution, and developed the computer software to perform numerical simulations. Using them, we analyzed the effects of each experimental parameter on the observed TCS effects by the simulations. The simulation studies indicated that: (i) the proton concentration in the solvent should be 10-30%, (ii) a larger pb, which is the bound fraction of the ligand, is preferred for higher saturation efficiency, (iii) the TCS method is applicable to systems where koff > 0.1 s−1, (iv) for koff ≥ 10 s−1, pb ≥ 0.1 is preferred, (v) for koff ∼ 1 s−1, pb ≥ 0.5 is preferred, and (vi) the TCS method is applicable to systems with large τc (∼1 μs), where pb is ∼0.01. The assumptions in the model spin simulation were experimentally verified, using the ubiquitin-YUH1 interaction. The established method will be useful for estimating and optimizing the TCS experimental conditions.