Quaternary structure of the neuronal protein NAP-22 in aqueous solution.
Quaternary structure of the neuronal protein NAP-22 in aqueous solution.
复制标题
水溶液中神经元蛋白 NAP-22 的四级结构。
DOI：
10.1016/s1570-9639(03)00191-2
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
S. Maekawa
中科院分区：
文献类型：
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作者：
R. Epand;E. Braswell;C. Yip;R. Epand;S. Maekawa
NAP-22是肉豆蔻酰化的阴离子蛋白，是神经元的洗涤剂不溶性部分的主要蛋白质组分。从膜中萃取后，它易溶于水。NAP-22将仅分配到具有特定脂质组合物的膜中。预期单体肉豆蔻酰化蛋白质不会具有脂质特异性。我们研究了NAP-22在溶液中的自缔合作用。沉降速度实验表明，蛋白质在很大程度上是相关的。低浓度极限s值为≤ 1.3 S，表明是高度不对称的单体。相比之下，一个nonmyristoylated形式的蛋白质显示没有证据的低聚化的速度沉降，并具有对应于NAP-22的最小组件的s值，但没有更高的低聚物的存在。沉降平衡运行表明，有一个快速可逆的平衡之间的单体和低聚体形式的蛋白质，然后由一个较慢的，更不可逆的协会成较大的聚集体。从新鲜制备的稀溶液中沉积在云母上的蛋白质的原位原子力显微镜显示云母表面上的二聚体。从沉降平衡数据获得的缔合常数的值表明，单体的重量浓度超过低于0.04 mg/ml的总蛋白质浓度的二聚体的重量浓度。由于NAP-22在发育中的大脑的神经元中的浓度是10.6mg/ml，如果蛋白质在溶液中，它将是寡聚体形式并且特异性地结合到富含胆固醇的结构域。我们证明，使用荧光共振能量转移，在低浓度下，NAP-22标记与得克萨斯红同样很好地结合到脂质体的磷脂酰胆碱，无论有或没有添加40摩尔%的胆固醇。因此，NAP-22的寡聚化有助于其在膜结合期间的脂质选择性。
NAP-22, a myristoylated, anionic protein, is a major protein component of the detergent-insoluble fraction of neurons. After extraction from the membrane, it is readily soluble in water. NAP-22 will partition only into membranes with specific lipid compositions. The lipid specificity is not expected for a monomeric myristoylated protein. We have studied the self-association of NAP-22 in solution. Sedimentation velocity experiments indicated that the protein is largely associated. The low concentration limiting s value is ∼1.3 S, indicating a highly asymmetric monomer. In contrast, a nonmyristoylated form of the protein shows no evidence of oligomerization by velocity sedimentation and has an s value corresponding to the smallest component of NAP-22, but without the presence of higher oligomers. Sedimentation equilibrium runs indicate that there is a rapidly reversible equilibrium between monomeric and oligomeric forms of the protein followed by a slower, more irreversible association into larger aggregates. In situ atomic force microscopy of the protein deposited on mica from freshly prepared dilute solution revealed dimers on the mica surface. The values of the association constants obtained from the sedimentation equilibrium data suggest that the weight concentration of the monomer exceeds that of the dimer below a total protein concentration of 0.04 mg/ml. Since the concentration of NAP-22 in the neurons of the developing brain is ∼0.6 mg/ml, if the protein were in solution, it would be in oligomeric form and bind specifically to cholesterol-rich domains. We demonstrate, using fluorescence resonance energy transfer, that at low concentrations, NAP-22 labeled with Texas Red binds equally well to liposomes of phosphatidylcholine either with or without the addition of 40 mol% cholesterol. Thus, oligomerization of NAP-22 contributes to its lipid selectivity during membrane binding.