Correlation between Glu194/Glu204 and the Schiff Base

re-lease group, Glu194 and Glu204, are located adverse and close to each other on the extracellular side of BR. In the [bR] state, Glu194/Glu204 and the retinal Schiff base are protonated. In the [M1] state, the Schiff base is deprotonated. In the [M2] state, Glu194/Glu204 is deprotonated, too. In the [N] and [O] state, the Schiff base is proto-nated again, while Glu194/Glu204 remains deprotoproto-nated. The photocycle and its proton transfer steps are shown in Figure 5.2 and the intermediate protonation states are listed in Table 5.1.

The correlation between both Glu194 and Glu204 and the retinal Schiff base is de-picted in Figure 5.24 for six BR structures. In general, the protonation behavior of Glu194/Glu204 and the retinal Schiff base is uncorrelated. However, interestingly a positive and also a negative correlation can be observed for Glu204 and the retinal Schiff base in L:1vjm.b. For M:1f4z and for N:1p8u, a small positive correlation can be seen for Glu194 or Glu204 and the retinal Schiff base at high pH values. For M:1kg8, the correla-tion between Glu194 and the retinal Schiff base is negative. In the following subseccorrela-tions, the correlation behavior is discussed in detail for the individual structures.

BR STRUCTURES

For bR:1c3w, the correlation coefficient of Glu194 or Glu204 and the retinal Schiff base fluctuates around 0 over the whole pH range. Similarly for bR:1f50, the correlation coefficient of Glu194 or Glu204 and the retinal Schiff base fluctuates around 0 over the whole pH range. The protonation probability of the retinal Schiff base is close to 1 over the complete pH range from 0 to 14 for both bR:1c3w and bR:1f50 (cf. Figure 5.11 a).

In bR:1c3w, the protonation probability of Glu194 is 0.99 at pH<12.5. Thereafter, it decreases slightly to 0.97 (cf. Figure 5.16 a). The protonation probability of Glu204 is 0.5 for the pH range of 0 to 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

5.5. Correlation of the Protonation Behavior 117

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 5.24. Correlation between Glu194/Glu204 and the Schiff base. The co-efficient of the correlation between both E194 and E204 and the Schiff base is plotted against pH for six BR structures.

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 short in bR:1c3w, Glu194 is protonated and Glu204 is deprotonated and in bR:1f50 the protonation is reversed. For bR:1c3w and bR:1f50, Glu194 or Glu204 and the retinal Schiff base are uncorrelated over the whole pH range.

L STRUCTURE

For L:1vjm.b, the correlation coefficient of Glu194 and the retinal Schiff base fluctuates around 0 at pH<4 and is exactly 0 thereafter. The correlation between Glu204 and the retinal Schiff base is 0 at pH = 0. It increases up to 0.25 at pH = 2 and then decreases to -0.35 at pH = 5.2 with a zero point at pH = 3.4. At pH = 7.5, the coefficient again ap-proximates 0 and at pH>8, it is exactly 0. As shown in Figure 5.11 c, at pH<2 the protonation probability of the retinal Schiff base is close to 1. In the pH range from 2 to 8, the Schiff base deprotonates. At pH>8, the protonation probability is close to 0 or exactly 0. Glu194 is nearly deprotonated in the pH range from 0 to 4. At pH>4, the protonation probability is exactly 0. The protonation probability of Glu204 is close to 1 at pH = 0. Then it decreases to approximately 0.1 at pH = 4 and approximately 0 at pH = 5. In the pH range from 8.5 to 14, Glu204 is completely deprotonated. The individ-ual probability curves of Glu194 and Glu204 are not shown. The sum of their protonation probabilities is depicted in Figure 5.15 c.

In the pH range from 4 to 14, Glu194 and in part the retinal Schiff base show no variance.

Their correlation coefficient is, therefore, 0. However, Glu194 and the retinal Schiff base are uncorrelated, too, when they show variance. In contrast, Glu204 and the Schiff base are slightly positively correlated in the pH range from 0 to 3.5, when both are mostly protonated. Then in the pH range from 3.5 to 7.5 , when Glu204 is mostly deprotonated and the Schiff base mostly protonated, they are slightly negatively correlated. At pH>8, the correlation coefficient is 0 due to the absent variance of Glu204.

M STRUCTURES

For M:1f4z, the correlation coefficient of Glu194 and the retinal Schiff base is approxi-mately 0 at pH<12. At high pH values the correlation increases slightly to 0.1 at pH = 14.

Similarly, the correlation coefficient of Glu204 and the retinal Schiff base is approxi-mately 0 at pH<12. In the pH range from 12 to 14 the correlation increases to about 0.3.

For M:1kg8, the correlation between Glu194 and the retinal Schiff base is exactly 0 at pH≤3 and approximately 0 up to pH 4.5. Furthermore at pH>10, the coefficient fluctuates around 0. However, in the pH range from 4.5 to 10, a negative correlation can be observed with a minimum of -0.5 at pH = 7.7. In contrast, the correlation between Glu204 and the retinal Schiff base is 0 over the whole pH range.

As shown in Figure 5.11 d, at pH<4 the protonation probability of the retinal Schiff base is close to 1. In the pH range from 4 to 8, the Schiff base deprotonates. In M:1f4z, the minimum protonation probability is approximately 0.15. At pH = 10, the protonation probability increases up to approximately 0.5 at pH = 14. In M:1kg8, the protonation probability is close to 0 in the pH range from 8 to 14. In M:1f4z, the protonation proba-bility 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 depro-tonates and at pH = 14, the protonation probability 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.

For M:1f4z, the behavior of Glu194 or Glu204 and the retinal Schiff base is slightly correlated at high pH values when both glutamate residues are mostly deprotonated and the Schiff base is partly protonated. The correlation between Glu204 and the Schiff base is more pronounced than between Glu194 and the Schiff base. For both Glu204 and the Schiff base the protonation probability is significantly higher in this pH range than for Glu194. Thus, the positive correlation may result from both residues being protonated at the same time.

For M:1kg8, Glu194 and the retinal Schiff base are negatively correlated when the first is still mostly protonated and the latter mostly deprotonated. At higher pH values, when both Glu194 and the Schiff base are virtually deprotonated their behavior is uncorrelated.

The correlation coefficient of Glu204 and the Schiff base is 0 over the whole pH range due to the absent variance of Glu204.

N STRUCTURE

For N:1p8u, the correlation coefficient of Glu194 and the retinal Schiff base is 0 at pH≤1.3 and fluctuates around 0 thereafter. Similarly, the correlation coefficient of Glu204 and the retinal Schiff base is 0 at pH≤1.3 and fluctuates around 0 thereafter.

However, at pH>10, the correlation coefficient increases to about 0.25 at pH = 14.

5.6. Implications for the Intermediate Structures 119