Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: Evaluation of reversible unfolding conditions
Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: Evaluation of reversible unfolding conditions
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DOI：
10.1021/bi9909039
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发表时间：
1999-11-16
期刊：
影响因子：
2.9
通讯作者：
Gouaux, E
中科院分区：
文献类型：
--
作者：
Chen, GQ;Gouaux, E
来自盐杆菌的细菌视紫红质 (sbR) 的野生型和突变型，由大肠杆菌过表达合成基因产生，在 1,2-二甲酰基-sn-甘油-3-磷酸胆碱 (DMPC)、3-[(3-胆酰胺丙基)二甲基氨基]-2-羟基-1-丙烷 (CHAPSO) 和十二烷基硫酸钠中可逆展开为了研究疏水表面极性残基取代对蛋白质稳定性的影响，将 G113Q、G116Q 突变体 [sbR(Q2)] 的去折叠行为与野生型 sbR [sbR(WT)] 进行比较，平衡条件下 sbR(Q2) 比 sbR(WT) 对 SDS 诱导的去折叠更敏感，动力学实验表明 sbR(Q2)比 sbR(WT) 对酸诱导的变性和热解折叠更敏感。由于 sbR(Q2) 中的突变发生在 sbR 去污剂嵌入的疏水表面上，因此这些突变造成的蛋白质不稳定支持了膜嵌入片段对于 sbR 稳定性很重要的概念。我们的实验通过评估 DMPC/CHAPSO/SDS 混合胶束中的可逆折叠和解折叠条件，为研究 sbR 的热力学稳定性提供了基础。
Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium salinarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1,2-dimristoyl-sn-glycero-3-phosphocholine (DMPC), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles, To study the effect on protein stability by substitutions on the hydrophobic surface with polar residues, the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)], sbR(Q2) was more sensitive to SDS-induced unfolding than sbR(WT) under equilibrium conditions, and kinetic experiments showed that sbR(Q2) was more sensitive to acid-induced denaturation and thermal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the detergent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are important for the stability of sbR. Our experiments provide the basis for studying the thermodynamic stability of sbR by evaluating reversible folding and unfolding conditions in DMPC/CHAPSO/SDS mixed micelles.