Folding kinetics of an α helical membrane protein in phospholipid bilayer vesicles
Folding kinetics of an α helical membrane protein in phospholipid bilayer vesicles
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DOI：
10.1016/j.jmb.2004.07.040
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发表时间：
2004-09-24
影响因子：
5.6
通讯作者：
Booth, PJ
中科院分区：
文献类型：
--
作者：
Allen, SJ;Curran, AR;Booth, PJ
我们报告了一个详细的动力学研究的折叠的a-螺旋膜蛋白在脂质双层环境。SDS变性的细菌视紫红质通过停流混合直接折叠成磷脂酰胆碱脂质囊泡。通过蛋白质荧光的时间分辨变化以及视网膜发色团的吸收，用毫秒时间分辨率监测折叠动力学。的动力学是类似于先前报道的折叠细菌视紫红质在洗涤剂或脂质胶束，除了存在一个额外的载脂蛋白中间体。我们认为，这个中间体是一个更大的内部二维压力存在于这些脂质囊泡胶束相比的结果。这些结果奠定了基础，为未来的研究，旨在了解的机制起源的影响，脂双层性质的蛋白质折叠。此外，使用生物学相关的磷脂酰胆碱脂质，以及直接快速混合过程来启动折叠反应，意味着该方法是普遍适用的，因此为改善跨膜α-螺旋蛋白体外折叠的理解铺平了道路。(C)2004 Elsevier Ltd.保留所有权利。
We report a detailed kinetic study of the folding of an a-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane a-helical proteins. (C) 2004 Elsevier Ltd. All rights reserved.