Johnson J E, Cornell R B
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Biochemistry. 1994 Apr 12;33(14):4327-35. doi: 10.1021/bi00180a029.
A peptide corresponding to a portion of the amphipathic alpha-helical region of CTP:phosphocholine cytidylyltransferase was synthesized. This region of the enzyme was proposed to be the membrane-binding domain [Kalmar, G.B., Kay, R.J., Lachance, A., Aebersold, R., & Cornell, R.B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6029]. We have shown that the peptide is physically associated with PG vesicles. CD of the peptide in buffer suggested a primarily random structure, while, in the presence of trifluoroethanol, the peptide was alpha-helical. Anionic lipid vesicles promoted an alpha-helical conformation, whereas neutral or cationic lipid vesicles did not alter the random structure of the peptide, suggesting a selective stabilization of the alpha-helix by anionic membranes. The fluorescence of the single tryptophan residue, which lies on the hydrophobic face of the amphipathic alpha-helix, was studied. Anionic lipid vesicles specifically induced a shift in the fluorescence to a lower wavelength. Fluorescence quenching by the aqueous-phase quencher, I-, and the lipid-phase quencher 9,10-dibromo-PC was used to determine the accessibility of the tryptophan to each of these environments. The presence of anionic lipid vesicles, but not nonanionic lipid vesicles, decreased the quenching by I- suggesting that, in the presence of anionic lipids, the tryptophan residue is poorly accessible to the aqueous I-. Dibromo-PC significantly quenched the fluorescence only when present in anionic vesicles, confirming the membrane location of the tryptophan residue and the lipid specificity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)
合成了一段与CTP：磷酸胆碱胞苷转移酶两亲性α-螺旋区域一部分相对应的肽段。该酶的这一区域被认为是膜结合结构域[卡尔马尔，G.B.，凯，R.J.，拉尚斯，A.，艾伯索尔德，R.，& 康奈尔，R.B.（1990年）《美国国家科学院院刊》87，6029]。我们已表明该肽段与磷脂酰甘油（PG）囊泡存在物理关联。该肽段在缓冲液中的圆二色性（CD）表明其主要为无规结构，而在三氟乙醇存在的情况下，该肽段呈α-螺旋结构。阴离子脂质囊泡促进α-螺旋构象形成，而中性或阳离子脂质囊泡不会改变该肽段的无规结构，这表明阴离子膜对α-螺旋具有选择性稳定作用。对位于两亲性α-螺旋疏水面上的单个色氨酸残基的荧光进行了研究。阴离子脂质囊泡特异性地诱导荧光向较低波长偏移。利用水相淬灭剂I⁻和脂相淬灭剂9,10 - 二溴磷脂酰胆碱（9,10 - dibromo - PC）进行荧光淬灭，以确定色氨酸在每种环境中的可及性。阴离子脂质囊泡的存在而非非阴离子脂质囊泡的存在降低了I⁻引起的淬灭，这表明在阴离子脂质存在的情况下，色氨酸残基对水相中的I⁻难以接近。仅当9,10 - 二溴磷脂酰胆碱存在于阴离子囊泡中时，它才会显著淬灭荧光，这证实了色氨酸残基的膜定位以及这种相互作用的脂质特异性。（摘要截短于250字）
