Back to Analysis of Direct and Reverse Titrations

Intermediate tight/-off rate binding, fast isomerization

Here we will compare outcomes of the four alternative models for conditions when binding is moderately tight and the off-rate is intermediate. The isomerization is chosen to be fast here. I choose binding affinity of 10⁷ M^-1 and off-rate constant of 50 s^-1. I choose separation between the major peaks of 100 s^-1 to generate most similar spectral appearance. Isomerization equilibrium is shifted 5:1 toward *-forms. The reverse isomerization rate is 170 s^-1to make k_ex=1000 s^-1.

Location: Ai_Bf/


Simulate setup U_Ai	Simulate setup U_R_Ai_Bf	Simulate setup U_L_Ai_Bf	Simulate setup U_RL_Ai_Bf




Fitting with the 2-site model:

Optimum norm: 6.81e-04 [1] Kd: 8.75973e-08 +/- 5.2e-09 [2] Koff: 5.04841e+01 +/- 1.5e-01 [3] Scale Factor: 2.05816e+00 +/- 1.4e-03	Optimum norm: 1.67e-02 [1] Kd: 2.93019e-07 +/- 3.7e-08 [2] Koff: 4.22543e+01 +/- 5.9e-01 [3] Scale Factor: 2.05051e+00 +/- 6.7e-03	Optimum norm: 8.24e-04 [1] Kd: 4.14096e-07 +/- 1.1e-08 [2] Koff: 5.00697e+01 +/- 1.6e-01 [3] Scale Factor: 2.05814e+00 +/- 1.5e-03	Optimum norm: 2.16e-03 [1] Kd: 1.56272e-08 +/- 4.5e-09 [2] Koff: 8.38918e+00 +/- 6.2e-02 [3] Scale Factor: 2.06848e+00 +/- 2.5e-03 The end of titration is systematically off
Simulate 2-site data for these fitted parameters
Simulate setup U_Ai_fit	Simulate setup U_Ai_fit_U_R	Simulate setup U_Ai_fit_U_L	Simulate setup U_Ai_fit_U_RL
Compare fitted and actual peak positions and line widths Comparisons/
plotdatasets 'U_Ai_fit_chem_shift' 'L/R' 'Frequency, /s'	plotdatasets 'U_R_Ai_Bf_fit_chem_shift' 'L/R' 'Frequency, /s'	plotdatasets 'U_L_Ai_Bf_fit_chem_shift' 'L/R' 'Frequency, /s'	plotdatasets 'U_RL_Ai_Bf_fit_chem_shift' 'L/R' 'Frequency, /s'
plotdatasets 'U_Ai_fit_LW' 'L/R' 'Frequency, /s'	plotdatasets 'U_R_Ai_Bf_fit_LW' 'L/R' 'Frequency, /s'	plotdatasets 'U_L_Ai_Bf_fit_LW' 'L/R' 'Frequency, /s'	plotdatasets 'U_RL_Ai_Bf_fit_LW' 'L/R' 'Frequency, /s'

Recalculation with the better sampling of the transition region L/R=0.85-1.05. Let' redo the calculation with a new setup file:


Simulate setup2 U_Ai	Simulate setup2 U_R_Ai_Bf	Simulate setup2 U_L_Ai_Bf	Simulate setup2 U_RL_Ai_Bf




The two-site exchange model will look the same in direct and reverse titration	Forward titration and reverse titrations look the same. However, the line peak shifts and line shapes are different. Need to fit with the 2-site model to see the effect