A designed cavity in the hydrophobic core of a four-alpha-helix bundle improves volatile anesthetic binding affinity.
A designed cavity in the hydrophobic core of a four-alpha-helix bundle improves volatile anesthetic binding affinity.
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DOI：
10.1021/bi9721290
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发表时间：
1998-01
期刊：
影响因子：
2.9
通讯作者：
J. Johansson;B. Gibney;F. Rabanal;K. Reddy;P. Dutton
中科院分区：
文献类型：
--
作者：
J. Johansson;B. Gibney;F. Rabanal;K. Reddy;P. Dutton
挥发性麻醉剂的蛋白质结合位点的结构特征正在使用一个确定的模型系统进行探索，该模型系统由一个带有疏水核心的四个α-螺旋束支架组成。早期的工作已经证明，一个原型疏水核心能够结合挥发性麻醉剂氟烷。通过在原型(α2)2四α螺旋束中引入一个简单的空腔，用较小的丙氨酸取代六个核心亮氨酸，提出了设计一种改进的亲和力麻醉剂结合部位的探索性工作。与没有这种腔的类似束芯(Kd=3.1+/-0.4 mm)相比，存在这种腔的挥发性麻醉剂与所设计的束芯结合的亲和力(Kd=0.71+/-0.04 mm)增加了4.4倍。这表明，天然蛋白质上存在的这种包装缺陷很可能被体内挥发性全身麻醉剂所占据。用丙氨酸取代6个疏水核心亮氨酸残基导致折叠束的不稳定性为1.7-2.7kcal/mol丙氨酸，尽管丙氨酸取代的束仍然表现出高度的热力学稳定性，总的折叠构象增量gh2O=14.3+/-0.8kcal/mol。自旋标记MTSSL与丙氨酸取代的四个α-螺旋束中的半胱氨酸残基的共价结合表明，双α-螺旋多肽以反方向二聚。四个α-螺旋束的旋转相关时间为8.1+/-0.5 ns，与早期对类似多肽的工作一致。荧光、远紫外圆二色谱和傅里叶变换红外光谱证实了色氨酸和半胱氨酸残基的疏水中心位置，实验结果与设计吻合较好。这些人工合成的小蛋白可能有助于研究小分子结合位点的结构特征。
The structural features of protein binding sites for volatile anesthetics are being explored using a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Earlier work has demonstrated that a prototype hydrophobic core is capable of binding the volatile anesthetic halothane. Exploratory work on the design of an improved affinity anesthetic binding site is presented, based upon the introduction of a simple cavity into a prototype (alpha 2)2 four-alpha-helix bundle by replacing six core leucines with smaller alanines. The presence of such a cavity increases the affinity (Kd = 0.71 +/- 0.04 mM) of volatile anesthetic binding to the designed bundle core by a factor of 4.4 as compared to an analogous bundle core lacking such a cavity (Kd = 3.1 +/- 0.4 mM). This suggests that such packing defects present on natural proteins are likely to be occupied by volatile general anesthetics in vivo. Replacing six hydrophobic core leucine residues with alanines results in a destabilization of the folded bundle by 1.7-2.7 kcal/mol alanine, although the alanine-substituted bundle still exhibits a high degree of thermodynamic stability with an overall folded conformational delta GH2O = 14.3 +/- 0.8 kcal/mol. Covalent attachment of the spin label MTSSL to cysteine residues in the alanine-substituted four-alpha-helix bundle indicates that the di-alpha-helical peptides dimerize in an anti orientation. The rotational correlation time of the four-alpha-helix bundle is 8.1 +/- 0.5 ns, in line with earlier work on similar peptides. Fluorescence, far-UV circular dichroism, and Fourier transform infrared spectroscopies verified the hydrophobic core location of the tryptophan and cysteine residues, showing good agreement between experiment and design. These small synthetic proteins may prove useful for the study of the structural features of small molecule binding sites.