Role of a conserved salt bridge between the PAS core and the N-terminal domain in the activation of the photoreceptor photoactive yellow protein
Role of a conserved salt bridge between the PAS core and the N-terminal domain in the activation of the photoreceptor photoactive yellow protein
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DOI：
10.1529/biophysj.107.106633
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发表时间：
2007-09-01
影响因子：
3.4
通讯作者：
Heyn, Maarten P.
中科院分区：
文献类型：
--
作者：
Hoersch, Daniel;Otto, Harald;Heyn, Maarten P.
用时间分辨吸收光谱和荧光光谱研究了离子强度对光感受器PYP的I-2中间体和信号态I-2 '之间构象平衡的影响以及对恢复到暗态的速率的影响。随着盐浓度增加至接近600 mM，回收率k（3）降低，I-2/I-2 '平衡（K）向I-2 '方向移动.在较高的离子强度下，两种效应都逆转。用一价（KCl、NaBr）和二价（MgCl 2、MgSO 4）盐的实验表明，低盐效应取决于离子强度，而不是阳离子或阴离子种类。这些观察结果可以通过考虑域间盐桥的活度系数在整个离子强度范围内进行描述。在低离子强度下，由于反离子屏蔽，活度系数降低，而在高离子强度下，盐与水的结合导致活度系数增加。从log k3和log K对离子强度平方根的曲线的初始斜率，发现相互作用基团的电荷的乘积为-1.3 +/-0.2，表明一价离子对。连接PAS核心的β-折叠和N-末端结构域的保守盐桥K110/E12是该离子对的主要候选者。为了检验这一假设，制备了突变体K110 A和E12 A。在K110 A中，I-2/ I-2 ′平衡的盐依赖性被消除，并且回收率大大降低到低于600 mM。此外，在低盐下，回收率比野生型慢6倍。在E12 A中，仍然存在显著的盐依赖性，这归因于K110和E9之间形成新的盐桥。在高盐逆转发生在这两个突变体表明，盐析稳定更紧凑的I2结构。然而，离液阴离子如SCN使I 2/I-2 '平衡向部分未折叠的I-2 '形式移动。盐键K110/E12在黑暗中使光感受器稳定在非活性状态，并在光诱导形成的信号传导状态中被破坏，允许N-末端结构域从β-支架PAS核心分离。
The effect of ionic strength on the conformational equilibrium between the I-2 intermediate and the signaling state I-2 ' of the photoreceptor PYP and on the rate of recovery to the dark state were investigated by time-resolved absorption and fluorescence spectroscopy. With increasing salt concentration up to similar to 600 mM, the recovery rate k(3) decreases and the I-2/I-2 ' equilibrium (K) shifts in the direction of I-2 '. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl2, MgSO4) salts show that the low salt effect depends on the ionic strength and not on the cation or anion species. These observations can be described over the entire ionic strength range by considering the activity coefficients of an interdomain salt bridge. At low ionic strength the activity coefficient decreases due to counterion screening whereas at high ionic strength binding of water by the salt leads to an increase in the activity coefficient. From the initial slopes of the plots of log k3 and log K versus the square root of the ionic strength, the product of the charges of the interacting groups was found to be - 1.3 +/- 0.2, suggesting a monovalent ion pair. The conserved salt bridge K110/E12 connecting the beta-sheet of the PAS core and the N-terminal domain is a prime candidate for this ion pair. To test this hypothesis, the mutants K110A and E12A were prepared. In K110A the salt dependence of the I-2/ I-2 ' equilibrium was eliminated and of the recovery rate was greatly reduced below; similar to 600 mM. Moreover, at low salt the recovery rate was six times slower than in wild-type. In E12A significant salt dependence remained, which is attributed to the formation of a novel salt bridge between K110 and E9. At high salt reversal occurs in both mutants suggesting that salting out stabilizes the more compact I2 structure. However, chaotropic anions like SCN shift the I2/I-2 ' equilibrium toward the partially unfolded I-2 ' form. The salt linkage K110/E12 stabilizes the photoreceptor in the inactive state in the dark and is broken in the lightinduced formation of the signaling state, allowing the N-terminal domain to detach from the beta-scaffold PAS core.