Saab Marie-belle, Estephan Elias, Cloitre Thierry, Legros René, Cuisinier Frédéric J G, Zimányi László, Gergely Csilla
Groupe d'Etude des Semi-conducteurs, UMR 5650, CNRS-Universite Montpellier II, 34095, Montpellier Cedex 5, France.
Langmuir. 2009 May 5;25(9):5159-67. doi: 10.1021/la9002274.
The membrane protein bacteriorhodopsin in its native membrane bound form (purple membrane) was adsorbed and incorporated into polyelectrolyte multilayered films, and adsorption was in situ monitored by optical waveguide light-mode spectroscopy. The formation of a single layer or a double layer of purple membranes was observed when adsorbed on negatively or positively charged surfaces, respectively. The purple membrane patches adsorbed on the polyelectrolyte multilayers were also evidenced by atomic force microscopy images. The driving forces of the adsorption process were evaluated by varying the ionic strength of the solution as well as the purple membrane concentration. At high purple membrane concentration, interpenetrating polyelectrolyte loops might provide new binding sites for the adsorption of a second layer of purple membranes, whereas at lower concentrations only a single layer is formed. Negative surfaces do not promote a second protein layer adsorption. Driving forces other than just electrostatic ones, such as hydrophobic forces, should play a role in the polyelectrolyte/purple membrane layering. The subtle interplay of all these factors determines the formation of the polyelectrolyte/purple membrane matrix with a presumably high degree of orientation for the incorporated purple membranes, with their cytoplasmic, or extracellular side toward the bulk on negatively or positively charged polyelectrolyte, respectively. The structural stability of bacteriorhodopsin during adsorption onto the surface and incorporation into the polyelectrolyte multilayers was investigated by Fourier transform infrared spectroscopy in attenuated total reflection mode. Adsorption and incorporation of purple membranes within polyelectrolyte multilayers does not disturb the conformational majority of membrane-embedded alpha-helix structures of the protein, but may slightly alter the structure of the extramembraneous segments or their interaction with the environment. This high stability is different from the lower stability of the predominantly beta-sheet structures of numerous globular proteins when adsorbed onto surfaces.
膜蛋白细菌视紫红质以其天然的膜结合形式（紫膜）被吸附并掺入聚电解质多层膜中，吸附过程通过光波导光模式光谱进行原位监测。当分别吸附在带负电或正电的表面上时，观察到形成了单层或双层紫膜。聚电解质多层膜上吸附的紫膜斑块也通过原子力显微镜图像得到证实。通过改变溶液的离子强度以及紫膜浓度来评估吸附过程的驱动力。在高紫膜浓度下，相互渗透的聚电解质环可能为第二层紫膜的吸附提供新的结合位点，而在较低浓度下仅形成单层。负表面不会促进第二层蛋白质层的吸附。除了静电作用之外的其他驱动力，如疏水力，应该在聚电解质/紫膜分层中起作用。所有这些因素的微妙相互作用决定了聚电解质/紫膜基质的形成，其中掺入的紫膜可能具有高度的取向性，其细胞质侧或细胞外侧分别朝向带负电或正电的聚电解质本体。通过衰减全反射模式的傅里叶变换红外光谱研究了细菌视紫红质在吸附到表面并掺入聚电解质多层膜过程中的结构稳定性。聚电解质多层膜中紫膜的吸附和掺入不会干扰蛋白质膜嵌入α-螺旋结构的构象主体，但可能会略微改变膜外区段的结构或它们与环境的相互作用。这种高稳定性与许多球状蛋白质在吸附到表面时主要为β-折叠结构的较低稳定性不同。
