5.5 Correlation of the Protonation Behavior
5.5.6 Correlation between Asp85 and Glu194/Glu204
Asp85 is located on the extracellular side of the retinal Schiff base in the center of BR.
The residues of the proton release group, Glu194 and Glu204, are located adverse and close to each other on the extracellular side of BR. In the [bR] state, Asp85 is deproto-nated and Glu194/Glu204 binds one proton. In the [M1] state, Asp85 is protonated and Glu194/Glu204 still binds one proton. In the [M2], [N] and [O] state Asp85 remains pro-tonated, while Glu194/Glu204 are deprotonated. The photocycle and its proton transfer steps are depicted in Figure 5.2 and the intermediate protonation states are listed in Table 5.1.
The correlation between Asp85 and both Glu194 and Glu204 is depicted in Figure 5.23 for six BR structures. As can be seen, in the pH range from 3 to 12, the protonation
0 2 4 6 8 10 12 14
pH
-1 -0.5 0 0.5 1
c ij
bR:1c3w bR:1f50 L:1vjm.b M:1f4z M:1kg8 N:1p8u
Figure 5.23. Correlation between Asp85 and Glu194/Glu204.The coefficient of the correlation between Asp85 and both Glu194 and Glu204 is plotted against pH for six BR structures.
behavior of Asp85 and Glu194/Glu204 is uncorrelated. At pH<3, a negative correlation coefficient can be observed for Asp85 and Glu204 in bR:1c3w and for Asp85 and Glu194 in bR:1f4z. At pH>12, a small negative correlation can be observed for Asp85 and both Glu194 and Glu204 in M:1f4z. In the following subsections, the correlation behavior is discussed in detail for the individual structures.
BR STRUCTURES
For bR:1c3w, the coefficient of the correlation between Asp85 and Glu194 fluctuates around 0 over the whole pH range. The correlation between Asp85 and Glu204 is -0.7 at pH = 0. Then, the coefficient increases to 0 at pH = 3.5 and fluctuates around 0 thereafter.
For bR:1f50, the correlation between Asp85 and Glu194 is -0.35 at pH = 0. The coefficient increases to 0 at pH = 2.5. Thereafter, the correlation fluctuates around 0. The coefficient of the correlation between Asp85 and Glu204 fluctuates around 0 in the pH range from 0 to 12.5. Thereafter, the correlation coefficient is exactly 0 with one exception.
For both bR:1c3w and bR:1f4z, the protonation probability of Asp85 is slightly larger than 0.5 at pH = 0, but decreases rapidly with increasing pH as shown in Figure 5.12 a.
At pH>3, the protonation probability is close to 0 for both structures. In bR:1c3w, the protonation probability of Glu194 is 0.99 at pH<12.5 (cf. Figure 5.16 a). Thereafter, it decreases slightly to 0.97. The protonation probability of Glu204 is 0.5 at pH<1, decreases to 0 at pH = 4. and remains close to 0 up to pH = 14. In bR:1f50, the protonation probability of Glu194 is close to 0 over the whole pH range. The protonation probability of Glu204 is close to 1 in the pH range from 0 to 9.5. Then, the probability decreases to 0.15 at pH = 14. For bR:1f50, the individual probability curves of Glu194 and Glu204 are not shown. The sum of their protonation probabilities is depicted in Figure 5.15 a. In general, in bR:1c3w Glu194 is protonated and Glu204 is deprotonated and in bR:1f50 the protonation is reversed, while Asp85 is deprotonated in both structures.
In bR:1c3w, the protonation behavior of Asp85 and Glu194 is uncorrelated. The protona-tion of Asp85 and Glu204 is negatively correlated when both residues are protonated to some degree at low pH values. In bR:1f50, the protonation behavior of Asp85 and Glu194
5.5. Correlation of the Protonation Behavior 115 is slightly negatively correlated when Asp85 is protonated in a number of states at low pH values. The protonation behavior of Asp85 and Glu204 is uncorrelated. Thus, Asp85 and either Glu194 or Glu204 are uncorrelated if they are deprotonated.
L STRUCTURE
For L:1vjm.b, the correlation between Asp85 and both Glu194 and Glu204 is 0 over the whole pH range. In the pH range from 0 to 11, Asp85 is fully protonated (cf. Figure 5.12 c).
At pH>11, its protonation probability is slightly smaller than 1 at 17 pH values. Glu194 is basically deprotonated in the pH range from 0 to 4. At pH>4, the protonation prob-ability is exactly 0. The protonation probprob-ability of Glu204 is close to 1 at pH = 0. Then the protonation probability decreases to approximately 0.1 at pH = 4 and approximately 0 at pH = 5. In the pH range from 8.5 to 10, Glu204 is completely deprotonated. The individual probability curves of Glu194 and Glu204 are not shown. The sum of their pro-tonation probabilities is depicted in Figure 5.15 c. Due to the absent variation of Asp85, Glu194 or Glu204, the correlation between Asp85 and both Glu194 and Glu204 is 0.
M STRUCTURES
For M:1f4z, the correlation between Asp85 and Glu194 is 0 at pH≤3.2 and fluctuates around 0 thereafter. In the pH range from 12 to 14 a small negative correlation of about -0.1 can be seen. Similarly, the correlation between Asp85 and Glu204 is 0 at pH≤3.2 and fluctuates around 0 thereafter. In the pH range of 12 to 14, the negative correlation is more pronounced than for Asp85 and Glu194 with a correlation coefficient of -0.3 at pH = 14. For M:1kg8, the correlation between Asp85 and Glu194 is 0 at pH<4. Thereafter a minimal negative correlation coefficient can be seen. The coefficient of the correlation between Asp85 and Glu204 is 0 over the whole pH range.
As shown in Figure 5.12 d, at pH<3.5 the protonation probability of Asp85 is 1 for both M structures. At pH>3.5, Asp85 deprotonates. In M:1kg8, the protonation probability remains above 0.8, while in M:1f4z, the protonation probability decreases to about 0.6 at pH = 14. In M:1f4z, the protonation probability of Glu194 is close to 0 over the whole pH range (cf. Figure 5.16 c). The protonation probability of Glu204 is close to 1 in the pH range from 0 to 11. Then, Glu204 deprotonates and at pH = 14, the protonation proba-bility is 0.25. In M:1kg8, Glu194 is fully protonated at pH≤3. In the pH range from 3 to 7.5, the protonation probability decreases slightly to 0.95. Thereafter, Glu194 quickly deprotonates. In the pH range from 11.5 to 14, the probability is close to 0. Glu204 is fully deprotonated over the whole pH range. For M:1kg8, the individual probability curves of Glu194 and Glu204 are not shown. The sum of their protonation probabilities is depicted in Figure 5.15 d.
In M:1f4z, the protonation behavior of Asp85 and Glu194 is slightly negatively correlated when Asp85 is partly deprotonated at high pH values. Asp85 and Glu204 are negatively correlated, when both Asp85 and Glu204 are partly deprotonated at high pH values. In 1kg8, the protonation behavior of Asp85 and Glu194 is uncorrelated even when both show some variance. The correlation between Asp85 and Glu204 is 0 over the whole pH range due to the absent variation of Asp85 or Glu204.
N STRUCTURE
For N:1p8u, the correlation between Asp85 and Glu194 is 0 over the whole pH range with the exception of some values at pH≥13.5. Similarly, the correlation between Asp85 and Glu204 is 0 over the whole pH range with the exception of five values at pH≥13.5.
The protonation probability of Asp85 is 1 in the pH range from 0 to 13.4, the (cf. Fig-ure 5.12 e). At pH≥13.5 at five pH values, a probability of 0.99 is observed. The pro-tonation probability of Glu194 is close to 0 over the whole pH range. In contrast, the protonation probability of Glu204 is close to 1 at pH<8.5. In the pH range from 8.5 to 14, the protonation probability decreases to 0.25. For N:1p8u, the individual probability curves of Glu194 and Glu204 are not shown. The sum of their protonation probabilities is depicted in Figure 5.15 e. In general, the correlation between Asp85 and both Glu194 and Glu204 is 0 due to the absent variance of Asp85. However, at the five pH values where Asp85 does show a small variance the protonation behavior of Asp85 and both Glu194 and Glu204 is uncorrelated, too.