Thermodynamics of Mistic protein folding

利用化学变性剂进行的体外展开和再折叠研究极大地促进了我们对水溶性和β桶膜蛋白的折叠热力学和动力学的理解。然而，α-螺旋膜蛋白的情况并非如此，α-螺旋膜蛋白约占所有蛋白质的三分之一，占所有药物靶点的一半以上。我们已经确定了已知结构的α-螺旋膜蛋白的第一个例子，可以被变性剂完全和可逆地展开：Mistic，来自枯草芽孢杆菌的110个残基，四跨膜蛋白，从洗涤剂胶束或脂质囊泡中解离，并在尿素滴度时呈现出未折叠的单体状态。利用位点定向诱变、多维光谱和Φ-value分析，我们的目标是利用这种独特的特性来(i)建立不同的膜模拟系统（胶束、单束和囊泡）中膜蛋白稳定性的定量比较；（ii）在氨基酸水平上深入了解这种不寻常的膜蛋白自发地以不依赖于跨位点的方式自我插入脂质双分子层和生物膜的能力；（iii）创建一个单体水溶性Mistic变体，允许在模拟膜和水环境之间直接比较蛋白质折叠。

In vitro unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble and β-barrel membrane proteins. However, this is not the case for α-helical membrane proteins, which constitute about one third of all proteins and more than half of all drug targets. We have identified the first example of an α-helical membrane protein of known structure that can be unfolded completely and reversibly by a denaturant: Mistic, a 110-residue, four-span membrane protein from Bacillus subtilis, dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. Using site-directed mutagenesis, multidimensional spectroscopy, and Φ-value analysis, we aim to exploit this unique property to (i) establish a quantitative comparison of membrane-protein stability in different membrane-mimetic systems (micelles, bicelles, and vesicles); (ii) obtain insights at the amino acid level into the ability of this unusual membrane protein to self-insert spontaneously and in a translocon-independent way into lipid bilayers and biological membranes; and (iii) create a monomeric water-soluble Mistic variant that allows a direct comparison of protein folding between membrane-mimetic and aqueous environments.