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. Author manuscript; available in PMC: 2013 Aug 14.
Published in final edited form as: Biochemistry. 2012 Jul 31;51(32):6441–6457. doi: 10.1021/bi300744z

Figure 4.

Figure 4

Optical titrations and binding isotherms for 17EE and 17-click binding to CYP3A4. Installment of 1,2,3-TRZ fragment on the 17EE framework to yield 17-click induces a type I (17EE, top left) to type II (17-click, top right) equilibrium binding conversion. Titrations were conducted in the presence of 2 μM CYP3A4 (100mM KPi + 20% glycerol, pH=7.4; 20 °C).,+ 17EE (left), + 17-click (right). Calculated binding isotherm for 17EE (bottom left) fitted best to a sequential two-site binding model where the binding of the first 17EE equivalent is ‘spin silent,’ while the second ‘spin-audible’ equivalent binds with positive cooperativity. 17-click binding isotherm (bottom right) displays hyperbolic binding behavior with similar affinity to parent 17EE.