Non-detergent isolation of a cyanobacterial photosystem I using styrene maleic acid alternating copolymers.
Non-detergent isolation of a cyanobacterial photosystem I using styrene maleic acid alternating copolymers.
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来自嗜热蓝细菌Thermosynechococcuselongatus（Te）的光系统I（PSI）是最大的膜蛋白复合物，其结构已通过X射线衍射解决。这种三聚体复合物具有36个蛋白质亚基，超过380个非共价结合的辅因子，分子量为1.2MDa。以前，它已被分离和表征的洗涤剂胶束使用非离子洗涤剂正十二烷基-β-d-麦芽糖苷（DDM）。我们现在已经成功地分离这种复杂的不使用洗涤剂，使用苯乙烯-马来酸（SMA）交替共聚物。有趣的是，发现部分酯化的共聚物制剂（SMA 1440，Cray Valley）在蓝藻类囊体膜中最有效。许多生物化学、生物物理学和功能测定已被应用于表征PSI的这种非去污剂形式，称为SMA脂质颗粒（SMALP）。与PSI-DDM相比，PSI-SMALP的沉降系数较低，表明密度降低或颗粒形状更长。我们发现PSI的77 K荧光最大值在PSI-SMALP中比PSI-DDM中红移，表明PsaA/B相关叶绿素的更天然的取向。我们报告，PSI-SMALPs的功能，尽管选择性损失的一个跨膜亚基，PsaF。这种损失可能反映了PSI核和PsaF的更不稳定的相互作用，或在共聚物插入和/或组装期间的选择性置换。PSI-SMALP与天然重组细胞色素c6的还原动力学降低，而非天然马心细胞色素c显示PSI-SMALP的还原速度快于PSI-DDM。这是最大的膜蛋白分离使用SMA共聚物，这项研究扩大了潜在的使用这种方法的大型超分子复合物的分离和表征。三聚体光系统I（PSI）从嗜热蓝细菌Thermosynechococcus elongatus（Te）是最大的膜蛋白复合物被封装在SMALP的日期。
Photosystem I (PSI) from the thermophilic cyanobacterium Thermosynechococcus elongatus (Te) is the largest membrane protein complex to have had its structure solved by X-ray diffraction. This trimeric complex has 36 protein subunits, over 380 non-covalently bound cofactors and a molecular weight of ∼1.2 MDa. Previously, it has been isolated and characterized in a detergent micelle using the non-ionic detergent n-dodecyl-β-d-maltoside (DDM). We have now succeeded in isolating this complex without the use of detergents, using styrene–maleic acid (SMA) alternating copolymer. Intriguingly, a partially esterified copolymer formulation (SMA 1440, Cray Valley) was found to be most efficient in cyanobacterial thylakoid membranes. A host of biochemical, biophysical and functional assays have been applied to characterize this non-detergent form of PSI, referred to as a SMA Lipid Particle (SMALP). The PSI-SMALP has a lower sedimentation coefficient compared to PSI-DDM, suggesting decreased density or a more extended particle shape. We show the 77 K fluorescence maximum for PSI is red shifted in PSI-SMALP compared to PSI-DDM, suggesting a more native orientation of PsaA/B associated chlorophyll. We report that PSI-SMALPs are functional despite the selective loss of one transmembrane subunit, PsaF. This loss may reflect a more labile interaction of the PSI core and PsaF, or a selective displacement during copolymer insertion and/or assembly. PSI-SMALP exhibited decreased reduction kinetics with native recombinant cytochromes c6, while non-native horse heart cytochrome c shows faster reduction of PSI-SMALP compared to PSI-DDM. This is the largest membrane protein isolated using SMA copolymers, and this study expands the potential use of this approach for the isolation and characterization of large supramolecular complexes. Trimeric Photosystem I (PSI) from the thermophilic cyanobacterium Thermosynechococcus elongatus (Te) is the largest membrane protein complex to be encapsulated within a SMALP to date.
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影响因子：
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DOI：
10.1007/s10969-016-9201-5
发表时间：
2016-03
期刊：
Journal of structural and functional genomics
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