CHARACTERIZATION OF 2 MEMBRANE-BOUND FORMS OF OMPA
CHARACTERIZATION OF 2 MEMBRANE-BOUND FORMS OF OMPA
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DOI：
10.1021/bi00006a013
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发表时间：
1995-02-14
期刊：
影响因子：
2.9
通讯作者：
TAMM, LK
中科院分区：
文献类型：
--
作者：
RODIONOVA, NA;TATULIAN, SA;TAMM, LK
通过限制性蛋白水解、偏振傅里叶变换红外光谱和荧光光谱研究了外膜蛋白A（OmpA）插入脂质双层的过程。在天然状态下，OmpA被认为形成了一个由八条反向平行的β链组成的桶。在本研究中，将其以展开形式分离、纯化并暴露于1-棕榈酰-2-油酰磷脂酰胆碱（POPC）、二肉豆蔻酰磷脂酰胆碱（DMPC）、二棕榈酰磷脂酰胆碱（DPPC）和三种在其sn-2链的不同位置溴化的磷脂（4，5-BrPC、9，10-BrPC和11，12-BrPC）的预制囊泡。有限的蛋白水解揭示了OmpA的两种膜结合形式，即"吸附"（35 kDa）和"插入"（30 kDa）形式[Surrey，T.，和Jahnig，F.等人（1992）Proc. Acad. sci. U.S.A.89，7457 - 7461]。在膜结合和重折叠后发现哪种形式取决于。取决于所用的脂质和温度。偏振衰减全反射（ATR）-FTIR光谱记录与OmpA绑定到锗支持的双层在这两种形式。酰胺I '带的位置表明两种膜结合形式的OmpA的β-结构的相当大的部分（吸附形式为35 - 45%，插入形式为45 - 55%）。插入形式的酰胺I '带的线性二色性的测量与反平行β-桶一致，其中链从膜法线倾斜约36度。β-链与双层法线的平均角度在35 kDa形式中可能大于插入形式。在溴化脂质的存在下，色氨酸荧光的淬灭程度取决于溴在脂肪酰基链中的位置。在插入形式中，4，5-BrPC最有效地淬灭荧光，这表明色氨酸残基的平均位置接近双层表面。在35 kDa的形式，色氨酸荧光最有效地淬灭9，10-BrPC，这表明这些残基的部分插入的疏水部分的双层。在可溶性猝灭剂丙烯酰胺的存在下的荧光光谱表明，在结合到膜后，OmpA的5个色氨酸残基在两种形式中以类似的程度被保护免受水性环境的影响。
The insertion of the outer membrane protein A (OmpA) into Lipid bilayers was studied by Limited proteolysis, polarized Fourier transform infrared (FTIR) spectroscopy, and fluorescence spectroscopy. In the native state, OmpA is thought to form a barrel of eight antiparallel beta-strands. For the present study, it was isolated in an,unfolded form, purified, and exposed to preformed vesicles of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and three phospholipids that were brominated in different positions of their sn-2 chains (4,5-BrPC, 9,10-BrPC, and 11,12-BrPC). Limited proteolysis revealed two membrane-bound forms of OmpA, namely an ''adsorbed'' (35 kDa) and an ''inserted'' (30 kDa) form [Surrey, T., and Jahnig, F. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7457-7461]. Which form was found after membrane binding and refolding depended. on the Lipids used and on the temperature. Polarized attenuated total reflection (ATR)-FTIR spectra were recorded with OmpA bound to germanium-supported bilayers in both forms. The position of the amide I' band indicated quite large fractions of beta-structure of OmpA in both membrane-bound forms (35-45% in the adsorbed form and 45-55% in the inserted form). Measurements of the linear dichroism of the amide I' bands in the inserted form are consistent with an antiparallel beta-barrel in which the strands are inclined at about 36 degrees from the membrane normal. The average angle of the beta-strands to the bilayer normal is likely larger in the 35 kDa form than in the inserted form. The extent of quenching of the tryptophan fluorescence in the presence of brominated lipids depended on the position of the bromines in the fatty acyl chain. In the inserted form, 4,5-BrPC quenched the fluorescence most efficiently, which indicates an average location of the tryptophan residues close to the bilayer surface. In the 35 kDa form, tryptophan fluorescence was most efficiently quenched by 9,10-BrPC, which indicates a partial insertion of these residues in the hydrophobic part of the bilayer. Fluorescence spectroscopy in the presence of the soluble quencher acrylamide showed that after binding to the membrane the five tryptophan residues of OmpA are protected from the aqueous environment to a similar extent in both forms.