de la Roche M A, Shen Z, Byers D M
Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
Arch Biochem Biophys. 1997 Aug 1;344(1):159-64. doi: 10.1006/abbi.1997.0203.
The amino acid sequence of Vibrio harveyi acyl carrier protein (ACP) is 86% identical to that of Escherichia coli ACP, although five nonconservative amino acid differences are concentrated in the loop region between helices I and II (residues 18-25). We have investigated the influence of these sequence differences on the hydrodynamic properties of the two ACPs and their fatty acylated derivatives. Hydropathy analysis suggests that V. harveyi ACP is more hydrophobic than E. coli ACP in the loop region, a prediction supported by stronger binding of V. harveyi acyl-ACPs (C12 to C16) to octyl-Sepharose. Gel filtration experiments indicated that both ACPs undergo a similar conformational expansion when pH was elevated from 7.5 (R(s) = 24 A) to 9.0 (R(s) = 30 A). Fatty acylation reversed this expansion: R(s) for 16:0-ACP was 12 A, independent of ACP source and pH. By contrast, V. harveyi and E. coli ACPs exhibited distinct gel electrophoretic properties. Fatty acylation of V. harveyi ACP produced a greater increase in mobility on a conformationally sensitive native gel system. Moreover, while both V. harveyi and E. coli ACPs migrated anomalously at 20 kDa on SDS-polyacrylamide gel electrophoresis, they exhibited strikingly different behavior on SDS gels upon acylation with longer chain fatty acids. These results indicate that E. coli and V. harveyi ACPs exhibit similar overall pH- and fatty acid-dependent conformational changes, but gel electrophoresis is more sensitive to structural differences due to variations of hydrophobicity and charge.
哈维氏弧菌酰基载体蛋白（ACP）的氨基酸序列与大肠杆菌ACP的氨基酸序列有86%的同源性，不过五个非保守氨基酸差异集中在螺旋I和螺旋II之间的环区（第18 - 25位残基）。我们研究了这些序列差异对两种ACP及其脂肪酰化衍生物流体动力学性质的影响。亲水性分析表明，在环区哈维氏弧菌ACP比大肠杆菌ACP更疏水，这一预测得到哈维氏弧菌酰基 - ACPs（C12至C16）与辛基 - 琼脂糖更强结合的支持。凝胶过滤实验表明，当pH从7.5（R(s)=24 Å）升高到9.0（R(s)=30 Å）时，两种ACP都会发生类似的构象扩张。脂肪酰化逆转了这种扩张：16:0 - ACP的R(s)为12 Å，与ACP来源和pH无关。相比之下，哈维氏弧菌和大肠杆菌的ACP表现出不同的凝胶电泳性质。在构象敏感的天然凝胶系统上，哈维氏弧菌ACP的脂肪酰化使其迁移率有更大增加。此外，虽然哈维氏弧菌和大肠杆菌的ACP在SDS - 聚丙烯酰胺凝胶电泳上均以20 kDa异常迁移，但在用长链脂肪酸酰化后，它们在SDS凝胶上表现出显著不同的行为。这些结果表明，大肠杆菌和哈维氏弧菌的ACP表现出相似的总体pH和脂肪酸依赖性构象变化，但凝胶电泳对由于疏水性和电荷变化导致的结构差异更敏感。
