Culham D E, Tripet B, Racher K I, Voegele R T, Hodges R S, Wood J M
Department of Microbiology and Guelph-Waterloo Centre for Graduate Work in Chemistry, University of Guelph, Guelph, Ontario, Canada.
J Mol Recognit. 2000 Sep-Oct;13(5):309-22. doi: 10.1002/1099-1352(200009/10)13:5<309::AID-JMR505>3.0.CO;2-R.
Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.
通过转运蛋白ProP对渗透保护剂的浓缩摄取有助于遭遇高渗培养基的大肠杆菌细胞的再水化。作为主要易化子超家族的一员，ProP在完整细菌、细胞质膜囊泡以及用纯化蛋白制备的蛋白脂质体中被渗透压升高激活。可溶性蛋白ProQ在体内对ProP的完全渗透激活也是必需的。ProP通过其渗透激活作用及其C端延伸区（预测形成α螺旋卷曲螺旋结构）与结构和功能同源物相区分。对应于ProP C端的合成多肽（ProP-p）形成了二聚体α螺旋卷曲螺旋结构。缺失26个C端氨基酸的转运蛋白ProP衍生物能够表达，但无活性。携带氨基酸变化K460I、Y467I和H495I（均位于核心卷曲螺旋结构的“a”位置）的衍生物比野生型转运蛋白需要更大的渗透压升高才能被激活。相同的变化扩展、稳定并改变了由ProP-p形成的卷曲螺旋结构的寡聚状态。氨基酸变化R488I（也位于“a”位置）进一步增加了激活ProP所需的渗透压升高幅度，降低了达到的活性并使ProP激活短暂。出乎意料的是，R488I替换使ProP-p形成的卷曲螺旋结构不稳定。螺旋不稳定变化I474P对ProP的活性和渗透激活的抑制作用更强。这些数据表明，ProP的羧基末端结构域可以形成具有异常性质的同二聚体α螺旋卷曲螺旋结构。它们表明C端结构域参与了ProP的渗透激活。
