Schenkl Selma, Portuondo Erwin, Zgrablic Goran, Chergui Majed, Suske Winfried, Dolder Max, Landau Ehud M, Haacke Stefan
Institut de Physique de la Matière Condensée, Université de Lausanne, 1015, Lausanne, Switzerland.
J Mol Biol. 2003 Jun 13;329(4):711-9. doi: 10.1016/s0022-2836(03)00508-4.
Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from proteins in different protonation states.
本文展示了从脂质立方相中收获的三维细菌视紫红质晶体的吸收光谱、荧光光谱和激发光谱。在室温、可控pH值和完全外部水合条件下进行的光谱实验相结合，揭示了三种不同蛋白质种类的存在。除了在紫膜中观察到的众所周知的形式外，我们还发现了另外两种种类，其相对贡献高达30%。由于这些光谱与含有细菌视紫红质的脱水或去离子化膜的光谱相似，我们认为位于视黄醛发色团附近的氨基酸残基改变了它们的质子化状态。我们提出结晶过程中和/或室温条件下的部分脱水是这种异质性的主要来源。这一论断得到了一项实验的支持，该实验表明在故意脱水后这些种类会相互转化，同时也得到了晶体学数据的支持，这些数据表明三维晶体中的面内晶胞与脱水细菌视紫红质膜的晶胞相当。在脱水结晶过程后蛋白质的完全水合受到阻碍。我们认为这种受阻的水扩散主要源于脂质双层对疏水晶体表面的封闭。由于其能够定量测定不同质子化状态蛋白质导致的组成异质性，目前的光谱学工作补充了晶体学数据。
