Ruan K H, Li D, Ji J, Lin Y Z, Gao X
Department of Internal Medicine, University of Texas Health Science Center, Houston 77030, USA.
Biochemistry. 1998 Jan 20;37(3):822-30. doi: 10.1021/bi971881y.
Thromboxane A2 synthase (TXAS) has been proposed to have two membrane-bound regions located in the NH2-terminal domain [Ruan, K.-H., Wang, L.-H., Wu, K. K., and Kulmacz, R. J. (1993) J. Biol. Chem, 268, 19483-19489; Ruan, K.-H., Li, P., Kulmacz, J. R., and Wu, K. K. (1994) J. Biol. Chem, 269, 20938-20942]. To test this hypothesis, a solution structure in membrane mimetic environments of a synthetic peptide corresponding to the second region of the NH2-terminal domain (TXAS residues 33-60) has been investigated by circular dichroism (CD), 2D nuclear magnetic resonance (NMR) spectroscopy, and peptidoliposome reconstitution. CD spectroscopy indicated that the peptide adopted a structure with significant alpha-helical content in 30% trifluoroethanol (TFE) or in dodecylphosphocholine (DPC) micelles, which mimic hydrophobic membrane environment. Through a combination of 2D NMR experiments in the presence of TFE or DPC micelles, complete 1H NMR assignments of the peptide have been obtained and the structure of the peptide has been determined. NH2-terminal segment of the peptide takes on a well-defined alpha-helical conformation; the center segment of the peptide, containing three prolines, adopts a bent conformation, and the C-terminal segment of the peptide exists in a mixture of rapidly interconverting conformations. These results provide direct structural evidence that residues 33-60 of the TXAS NH2-terminal domain contain a second membrane anchor region, with at least residues 35-46 having their helical structure expected for hydrophobic interaction with the membrane. The orientation of the peptide in DPC micelles was evaluated from the effect of incorporation of a spin-label 12-doxylstearate into the micelles. The peptide portions, found to be immersed in the micelles, include the helical segment, the bent segment, and some hydrophobic residues within the C-terminal segment. Two additional synthetic peptides, one corresponding to the NH2-terminal helical segment (TXAS residues 33-46) and the other including the bent and the C-terminal segments (TXAS residues 47-60) were analyzed for their ability to incorporate into peptidoliposomes. The helical peptide readily incorporated into liposomes; the other peptide did not. These results support the presence of a second functional membrane anchor region localized to the helical segment within TXAS residues 33-46, with passive membrane contacts in the bent and the C-terminal segments of the peptide (TXAS residues 47-60) due to immersion of the helical in the membrane.
血栓素A2合酶（TXAS）被认为在其氨基末端结构域有两个膜结合区域[阮，K.-H.，王，L.-H.，吴，K. K.，以及库尔马茨，R. J.（1993年）《生物化学杂志》，268卷，第19483 - 19489页；阮，K.-H.，李，P.，库尔马茨，J. R.，以及吴，K. K.（1994年）《生物化学杂志》，269卷，第20938 - 20942页]。为验证这一假设，通过圆二色性（CD）、二维核磁共振（NMR）光谱以及肽脂质体重构技术，对对应于氨基末端结构域第二个区域（TXAS第33 - 60位残基）的合成肽在膜模拟环境中的溶液结构进行了研究。CD光谱表明，该肽在模拟疏水膜环境的30%三氟乙醇（TFE）或十二烷基磷酸胆碱（DPC）胶束中呈现出具有显著α - 螺旋含量的结构。通过在TFE或DPC胶束存在下进行二维NMR实验的组合，已获得该肽的完整1H NMR归属并确定了其结构。该肽的氨基末端片段呈现出明确的α - 螺旋构象；肽的中心片段含有三个脯氨酸，呈弯曲构象，而肽的羧基末端片段以快速相互转换的构象混合物形式存在。这些结果提供了直接的结构证据，表明TXAS氨基末端结构域的第33 - 60位残基包含第二个膜锚定区域，其中至少第35 - 46位残基具有预期与膜进行疏水相互作用的螺旋结构。从将自旋标记物12 - 硬脂酰氧基硬脂酸酯掺入胶束的效果评估了该肽在DPC胶束中的取向。发现浸入胶束中的肽段包括螺旋段、弯曲段以及羧基末端片段内的一些疏水残基。另外分析了两个合成肽，一个对应于氨基末端螺旋段（TXAS第33 - 46位残基），另一个包括弯曲段和羧基末端段（TXAS第47 - 60位残基），以研究它们掺入肽脂质体的能力。螺旋肽很容易掺入脂质体；另一个肽则不能。这些结果支持在TXAS第33 - 46位残基内的螺旋段存在第二个功能性膜锚定区域，由于螺旋段浸入膜中，肽的弯曲段和羧基末端段（TXAS第47 - 60位残基）与膜存在被动接触。
