Structure retention of silica gel-encapsulated bacteriorhodopsin in purple membrane and in lipid nanodiscs
Structure retention of silica gel-encapsulated bacteriorhodopsin in purple membrane and in lipid nanodiscs
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DOI：
10.1016/j.colsurfb.2019.110680
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发表时间：
2020-02-01
影响因子：
5.8
通讯作者：
Longo, Marjorie L.
中科院分区：
文献类型：
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作者：
Gakhar, Sukriti;Risbud, Subhash H.;Longo, Marjorie L.
在天然紫膜（BR-PM）和合成脂质纳米盘（BR nanodisc）环境中，将膜蛋白细菌视紫红质（BR）包封在溶胶-凝胶衍生的多孔硅胶整料中。通过将紫膜溶解在由两亲性苯乙烯-马来酸（SMA）共聚物稳定的盘状磷脂双层膜中，制备了BR纳米盘。紫外-可见吸收光谱和动态光散射表明形成了溶解在平均直径为10.2 +/-0.7 nm的脂质纳米盘中的BR单体。荧光和吸收光谱技术被用来探测构象，环境，和旋转的变化与色氨酸残基和共价结合的视网膜部分的BR截留后，在二氧化硅基质。我们发现，在两种膜环境中的固定BR保留其结合的视网膜辅因子和辅因子的能力，进行构象变化后，BR的活性作为质子转运蛋白所必需的光照。对于紫膜片段，结果表明，包封后BR周围孔中的局部pH对其在高于50 ℃的温度下的稳定性很重要。在相同的缓冲条件下，分别在80 ° C（没有构象变化）和50 ° C（有构象变化）开始从二氧化硅包封的BR-PM和BR纳米盘释放视黄醛，反映了蛋白质-蛋白质（三聚体与单体）和蛋白质-脂质相互作用的差异。
The integral membrane protein, bacteriorhodopsin (BR) was encapsulated in sol-gel derived porous silica gel monoliths in native purple membrane (BR-PM) and synthetic lipid nanodisc (BR nanodisc) environments. BR nanodiscs were synthesized by solubilizing purple membrane in discoidal phospholipid bilayer stabilized by amphipathic Styrene-Maleic Acid (SMA) copolymer. UV-vis absorbance spectroscopy and dynamic-light scattering indicated the formation of BR monomers solubilized in lipid nanodiscs 10.2 +/- 0.7 nm in average diameter. Fluorescence and absorbance spectroscopic techniques were utilized to probe conformational, environmental, and rotational changes associated with the tryptophan residues and the covalently-bound retinal moiety of BR upon entrapment in the silica matrix. We show that the immobilized BR in both membrane environments retained its bound retinal cofactor and the ability of the cofactor to undergo conformational changes upon light illumination necessary for BR's activity as a proton transporter. For purple membrane fragments, the results indicated that the local pH in the pores around BR after encapsulation was important for its stability at temperatures higher than 50 degrees C. Under the same buffering conditions, retinal was released from silica-encapsulated BR-PM and BR nanodiscs beginning at 80 degrees C (without a conformational change) and 50 degrees C (with a conformational change), respectively, reflecting differences in protein-protein (trimeric vs. monomeric) and protein-lipid interactions.