Mi Dehui, Kim Hak Jun, Hadziselimovic Arina, Sanders Charles R
Department of Biochemistry and Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8725, USA.
Biochemistry. 2006 Aug 22;45(33):10072-84. doi: 10.1021/bi060887x.
Escherichia coli diacylglycerol kinase (DAGK) is a homotrimeric helical integral membrane protein in which a number of single-site mutations to cysteine are known to promote misfolding. Here, effects of other amino acid replacements have been explored using a folding assay based on the dilution of acidic urea/DAGK stock solutions into detergent/lipid mixed micelles. DAGK with an I110P or I110R mutation in the third transmembrane helix could not be purified because its expression was toxic to the E. coli host, most likely because of severe folding defects. Other mutations at Ile110 enhanced irreversible misfolding to varying degrees that generally correlated both with the polarity of the inserted amino acid and with the degree of protein destabilization. However, the I110W mutant was an exception in that it was highly misfolding prone while at the same time being more stable than the wild-type protein. This contrasts with I110Y, which also exhibited enhanced stability but folded with an efficiency similar to that of the wild type. For most mutants, the critical step leading to irreversible misfolding occurred for monomeric DAGK prior to trimerization and independent of association with mixed micelles. Misfolding of DAGK evidently involves the formation of incorrect monomer tertiary structure. Mutations appear to enhance misfolding by disfavoring the formation of correct structure rather than by directly stabilizing the misfolded state. Finally, when urea-solubilized DAGK was diluted into detergent/lipid-free buffer, it retained a significant degree of folding competency over a period of minutes. This property may be relevant to membrane protein folding in cells under conditions where the usual machinery associated with membrane integration is saturated, dysregulated, or dysfunctional.
大肠杆菌二酰基甘油激酶（DAGK）是一种同三聚体螺旋整合膜蛋白，已知其中一些半胱氨酸单点突变会促进错误折叠。在此，使用基于将酸性尿素/DAGK储备溶液稀释到去污剂/脂质混合胶束中的折叠测定法，探索了其他氨基酸替换的影响。在第三个跨膜螺旋中具有I110P或I110R突变的DAGK无法纯化，因为其表达对大肠杆菌宿主有毒，很可能是由于严重的折叠缺陷。Ile110处的其他突变在不同程度上增强了不可逆的错误折叠，这通常与插入氨基酸的极性以及蛋白质不稳定程度相关。然而，I110W突变体是个例外，它极易发生错误折叠，同时比野生型蛋白更稳定。这与I110Y形成对比，I110Y也表现出增强的稳定性，但折叠效率与野生型相似。对于大多数突变体，导致不可逆错误折叠的关键步骤发生在三聚化之前的单体DAGK中，且与混合胶束的结合无关。DAGK的错误折叠显然涉及不正确的单体三级结构的形成。突变似乎通过不利于正确结构的形成而不是直接稳定错误折叠状态来增强错误折叠。最后，当将尿素溶解的DAGK稀释到不含去污剂/脂质的缓冲液中时，它在几分钟内仍保持相当程度的折叠能力。在与膜整合相关的正常机制饱和、失调或功能失调的条件下，这种特性可能与细胞中的膜蛋白折叠有关。
