Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support.
Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support.
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DOI：
10.1371/journal.pone.0151582
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Booth PJ
中科院分区：
文献类型：
--
作者：
Di Bartolo N;Compton EL;Warne T;Edwards PC;Tate CG;Schertler GF;Booth PJ
人们对定义整合膜蛋白正确折叠和稳定性的因素知之甚少。仅对少数选定的膜蛋白的折叠进行了仔细检查，这使得对大蛋白家族（如 G 蛋白偶联受体 (GPCR)）的了解存在相当大的缺陷。完全可逆折叠对于任何膜蛋白来说都是有问题的，但这一主导受体家族却无法实现。此外，从变性状态恢复受体的尝试效率低下，最多只能产生 40-70% 的功能蛋白。我们提出了一种原型家族成员β1-肾上腺素受体可逆解折叠的方法，与未经历任何解折叠并保留其原始折叠结构的受体相比，在配体结合方面实现了折叠功能状态100%的恢复。我们利用固体支持物上的重折叠，这可以避免批量解决方案中发生的不需要的相互作用和聚集。我们确定展开和重新折叠时结构和功能的变化。此外，我们采用了一种相对较新的膜蛋白折叠方法；脉冲蛋白水解。 β1-肾上腺素能受体的完全重折叠发生在正癸基-β-D-麦芽糖苷 (DM) 胶束中，从尿素变性状态开始，如恢复其原始螺旋结构、配体结合和蛋白质荧光所示。固体支持物上的成功重折叠策略为功能性 GPCR 和其他膜蛋白的受控重折叠和恢复提供了一种明确的方法，这些蛋白一旦从天然膜中分离出来，就会遭受不稳定和不可逆变性。
The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.