Yamazaki Y, Hatanaka M, Kandori H, Sasaki J, Karstens W F, Raap J, Lugtenburg J, Bizounok M, Herzfeld J, Needleman R
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Biochemistry. 1995 May 30;34(21):7088-93. doi: 10.1021/bi00021a021.
Fourier transform infrared spectra of the L intermediate of light-adapted bacteriorhodopsin were examined for recombinant proteins with amino acid substitutions at Thr46 and Asp96. Two O-H stretching vibrational bands of water, at 3607 and 3577 cm-1, change into stronger H-bonding states in L of the wild type. Thr46-->Val substitution abolished these bands in spite of the fact that [3-18O]threonine-labeling did not shift them, indicating that they correspond to coordination of the water with Thr46. The two water bands were restored, although with changed frequencies, by an additional Asp96-->Asn substitution in Thr46-->Val/Asp96-->Asn. A single Asp96-->Asn substitution abolished the 3607 cm-1 band. Thus, Asp96 also takes part in structural changes in water. The perturbations of these water molecules in the L intermediate displayed a weak correlation with the ratio of intensity change in the two vibrational bands of the Schiff base mode at 1312 and 1301 cm-1 and the rate for the deprotonation of the Schiff base at the L-to-M reaction of the photocycle. We find, therefore, that the water molecules in the cytoplasmic Asp96-Thr46 domain, which comprises the site of proton uptake after formation of the M intermediate, undergo structural changes in the L intermediate already. These changes are transmitted to the extracellular domain and affect interaction of the Schiff base with Asp85, that is far removed from this region.
对在Thr46和Asp96处有氨基酸替换的重组蛋白，检测了光适应细菌视紫红质L中间体的傅里叶变换红外光谱。野生型L中，水的两个O-H伸缩振动带，位于3607和3577 cm-1，转变为更强的氢键状态。尽管[3-18O]苏氨酸标记未使它们发生位移，但Thr46→Val替换消除了这些谱带，表明它们对应于水与Thr46的配位。在Thr46→Val/Asp96→Asn中额外的Asp96→Asn替换使这两个水谱带恢复，尽管频率发生了变化。单一的Asp96→Asn替换消除了3607 cm-1的谱带。因此，Asp96也参与了水的结构变化。L中间体中这些水分子的扰动与席夫碱模式在1312和1301 cm-1处的两个振动带强度变化的比率以及光循环中L到M反应时席夫碱去质子化的速率呈弱相关。因此，我们发现，细胞质Asp96-Thr46结构域中的水分子，该结构域包含M中间体形成后质子摄取的位点，在L中间体中已经发生了结构变化。这些变化传递到细胞外结构域，并影响席夫碱与远离该区域的Asp85的相互作用。
