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. 2009 Mar;133(3):263–282. doi: 10.1085/jgp.200810079

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© 2009 Zhang et al.

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Figure 12. — A model in which two N termini can dwell within the antechamber at a time may better account for the data. (A) The two left-hand columns of states correspond to Scheme 1 under conditions of low Po and no inactivation and, therefore, approximate closed-channel conditions for a model in which one N terminus can occupy the antechamber at a time. The additional states highlighted in the dotted rectangle propose that an additional N terminus can also bind, presumably in the antechamber, thereby being protected from digestion by trypsin. Red and black ovoids correspond to unprotected and protected N termini, respectively. (B) Both one- and two-site models were used to simulate the trypsin-mediated removal of inactivation over a range of values of P, the binding constant for the site producing closed-channel–associated protection. Fractional occupancy was calculated as: P_a = p_f/(p_f+p_r), where p_f and p_r are defined as the rates of binding and unbinding of an N terminus in the antechamber (Table II). Both models predict a similar prolongation of τ_d (filled circles) with occupancy within the antechamber, whereas the two differ in regards to the limiting power term at high fractional occupancies (open circles). Note that, for the one-site model, the relationship between fractional occupancy, prolongation of τ_d, and n were plotted incorrectly in our previous report (Zhang et al., 2006), as described in Materials and methods. (C) The prolongation of τ_d is plotted as a function of the power term for the one-site (red) and two-site (blue) models. The horizontal lines mark the range of prolongations, relative to the case for n = 4, expected for power terms in the range of 2 to 3. The filled black circles correspond to measurements of n and calculations of prolongation of τ_d for wild-type β2 and mutated β2 constructs, assuming that the digestion rate of β3a most closely reflects digestion of an N terminus without protection in the antechamber. If the rate of digestion of the W4G construct is used as the τ_d(min), the points are shifted leftward, but still favor the idea that two sites are involved in closed-channel–associated protection.