Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
J Biol Chem. 2010 Oct 1;285(40):30558-66. doi: 10.1074/jbc.M110.128298. Epub 2010 Jul 21.
Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca(2+) and Mg(2+) and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by (15)N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.
细菌酰基辅酶 A 蛋白 (ACP) 是一种高度带负电荷的 9 kDa 蛋白，作为脂肪酸生物合成的辅助蛋白发挥作用。大肠杆菌 ACP 在中性 pH 值和不存在二价阳离子的情况下折叠，而非常相似的 86%序列同一性的哈维氏弧菌 ACP 在相同条件下未折叠。V. harveyi ACP 在添加二价阳离子（如 Ca(2+) 和 Mg(2+)）后采用折叠构象，先前已经鉴定出 A75H 突变体，该突变体在不存在二价阳离子的情况下在 pH 7 下恢复折叠构象。在这项研究中，我们试图使用 NMR 光谱和生物物理方法了解 V. harveyi ACP 的独特折叠行为。V. harveyi ACP A75H 的 NMR 溶液结构显示出具有四个螺旋围绕疏水核心的典型 ACP 结构，带有一个与溶剂隔离的狭窄口袋以容纳酰基链。在中性 pH 值下带电荷的 His-75 与螺旋 IV 远 C 末端的 Tyr-71 参与堆积相互作用。ACP 的 pH 滴定和静电分布表明，V. harveyi ACP 由于螺旋 II 周围的阴离子电荷排斥而不稳定，该排斥可以部分被 His-75 中和，并进一步被二价阳离子结合所降低。这得到了差示扫描量热法数据的支持，该数据表明钙结合进一步将 V. harveyi ACP A75H 的熔点提高了约 20°C。二价阳离子结合不会改变 (15)N NMR 弛豫实验在 ps-ns 时间尺度上的 ACP 动力学，然而，它显然通过氢氘交换研究稳定了蛋白质折叠。最后，我们证明大肠杆菌 ACP H75A 突变体与野生型 V. harveyi ACP 一样未折叠，进一步强调了该特定残基对正确蛋白质折叠的重要性。
