Spruijt R B, Meijer A B, Wolfs C J, Hemminga M A
Wageningen University and Research Center, Department of Biomolecular Sciences, Laboratory of Molecular Physics, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
Biochim Biophys Acta. 2000 Dec 20;1509(1-2):311-23. doi: 10.1016/s0005-2736(00)00314-x.
During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17-24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.
在感染过程中，丝状噬菌体M13的主要衣壳蛋白位于宿主大肠杆菌的细胞质膜中。本研究聚焦于衣壳蛋白N端部分在膜结合状态下的构型特性。为此，在衣壳蛋白的第3、7、9、10、11、12、13、14、15、17、19、21、22、23和24位产生X-半胱氨酸取代，覆盖蛋白的N端部分。所有使用的衣壳蛋白突变体通过在大肠杆菌中过表达成功以毫克量产生。突变衣壳蛋白被标记并重构到磷脂混合双层中。使用连接到半胱氨酸SH基团的AEDANS作为荧光探针，从最大发射波长推导出标记位点周围局部环境的极性信息。通过测定荧光猝灭剂丙烯酰胺和5-硬脂酰氧基硬脂酸的可及性获得额外信息。通过采用由TFE和SDS提供的均匀衣壳蛋白环境，可以排除衣壳蛋白主链的局部效应或残基的极性。我们的数据表明，在脂质与蛋白比例约为100时，蛋白的N端臂从第3位残基逐渐进入膜内直至第19位残基。连接衣壳蛋白螺旋部分的铰链区（第17 - 24位残基）被发现更深入地嵌入膜中。一个或多个膜锚定氨基酸残基赖氨酸8、苯丙氨酸11和亮氨酸14的取代，导致蛋白N端部分重排成更伸展的构象。通过降低脂质与蛋白比例，使膜表面每个衣壳蛋白所占空间减小，N端臂也可被迫形成这种构象。发现在体内认为相关的范围内，磷脂头部基团组成对蛋白N端部分重排的影响可忽略不计。从我们的实验中我们得出结论，膜锚定和空间限制效应是衣壳蛋白N端部分在膜中进行结构重排的关键因素。
