DNA-Mediated Stack Formation of Nanodiscs.
DNA-Mediated Stack Formation of Nanodiscs.
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DOI：
10.3390/molecules26061647
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发表时间：
2021-03-16
期刊：
影响因子：
--
通讯作者：
Oertel J
中科院分区：
文献类型：
--
作者：
Subramanian M;Kielar C;Tsushima S;Fahmy K;Oertel J
载脂蛋白A-1衍生的膜支架蛋白（MSP）已成为产生用于膜蛋白研究的纳米级盘状膜模拟物（纳米盘）的通用工具。最近的努力旨在利用其控制的脂蛋白的比例和大小分布安排膜蛋白在规则的超分子结构的衍射研究。因此，直接膜蛋白结晶，这仍然是膜蛋白的结构确定的限制因素，将被规避。我们在这里描述的膜支架蛋白MSP1D1结合的纳米盘使用工程半胱氨酸的巯基反应性的多聚体的形成。选择MSP 1D1中的突变位置N42和K163以支持化学修饰，如通过用芘荧光标记所证明的。通过圆二色谱、差示光散射和尺寸排阻色谱证明了对纳米盘形成和结构的最小干扰。由MSP 1D1_N42C变体形成的纳米盘的直接二硫键形成导致具有低产率的二聚体和三聚体。相比之下，透射电子显微镜显示，寡核苷酸与MSP1D1的工程化半胱氨酸的连接允许通过携带互补链和柔性间隔区的纳米盘群体的杂交生长堆叠纳米盘的亚微米尺寸的束。
Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.
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影响因子：
16.6
作者：
Gisriel, Chris;Coe, Jesse;Zatsepin, Nadia A.
通讯作者：
Zatsepin, Nadia A.
DOI：
10.1073/pnas.062565599
发表时间：
2002-05-14
影响因子：
11.1
作者：
Bayburt, TH;Sligar, SG
通讯作者：
Sligar, SG
影响因子：
8.6
作者：
Guilbaud, Sebastien;Salome, Laurence;Tardin, Catherine
通讯作者：
Tardin, Catherine
影响因子：
3.4
作者：
Kopf, Adrian H.;Dorr, Jonas M.;Killian, J. Antoinette
通讯作者：
Killian, J. Antoinette
影响因子：
3.4
作者：
Blanchette, Craig D.;Cappuccio, Jenny A.;Sulchek, Todd A.
通讯作者：
Sulchek, Todd A.