Schobert B, Lanyi J K, Oesterhelt D
Department of Physiology and Biophysics, University of California, Irvine 92717.
EMBO J. 1988 Apr;7(4):905-11. doi: 10.1002/j.1460-2075.1988.tb02895.x.
Halorhodopsin (HR), the light-driven chloride pump in halobacteria, was digested with various proteolytic enzymes. As expected, carboxypeptidase A removed 14 amino acids from the C-terminal tail of detergent-solubilized HR, producing a fragment of 25.2 kd in size. Membrane-associated HR could be digested as well, but not in right-side-out sealed cell envelope vesicles. We conclude, therefore, that the orientation of HR in the cytoplasmic membrane is such that the C-terminal tail faces the cytoplasmic side. Tryptic digestion of detergent-solubilized HR resulted in the removal of the same C-terminal segment, but also in the production of two more cleavage products (molecular masses of 20.9 and 16.8 kd respectively). These cleavage sites were determined by amino acid sequencing of the newly produced N termini, and they turned out to be within interhelical loops in an earlier proposed structural model for HR. Incubation with chymotrypsin and thermolysin yielded different sites of cleavage, but also in regions which were proposed to be accessible on the surface of the protein. Since the results show that three of six proposed interhelical loop segments contain proteolytic digestion sites, they support the proposed structural model for HR.
嗜盐菌视紫红质（HR）是嗜盐菌中的光驱动氯离子泵，用各种蛋白水解酶进行消化。正如预期的那样，羧肽酶A从去污剂溶解的HR的C末端尾巴上除去了14个氨基酸，产生了一个大小为25.2 kd的片段。与膜相关的HR也可以被消化，但在外翻密封的细胞膜囊泡中则不能。因此，我们得出结论，HR在细胞质膜中的取向是C末端尾巴面向细胞质一侧。对去污剂溶解的HR进行胰蛋白酶消化，不仅导致相同的C末端片段被去除，还产生了另外两种裂解产物（分子量分别为20.9和16.8 kd）。这些裂解位点通过对新产生的N末端进行氨基酸测序来确定，结果表明它们位于先前提出的HR结构模型的螺旋间环内。用胰凝乳蛋白酶和嗜热菌蛋白酶孵育产生了不同的裂解位点，但也在蛋白质表面被认为可及的区域。由于结果表明六个提出的螺旋间环段中有三个包含蛋白水解消化位点，因此它们支持所提出的HR结构模型。
