Lorch Mark, Booth Paula J
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
J Mol Biol. 2004 Dec 3;344(4):1109-21. doi: 10.1016/j.jmb.2004.09.090.
Membrane protein folding has suffered from a lack of detailed kinetic studies, particularly with regard to the insertion of denatured protein into lipid bilayers. We present a detailed in vitro kinetic study of the association of a denatured, transmembrane alpha helical protein with lipid vesicles. The mechanism of folding of Escherichia coli diacylglycerol kinase from a partially denatured state in urea has been investigated. The protein associates with lipid vesicles to give a protein, vesicle complex with an apparent association constant of 2 x 10(6) M(-1) s(-1). This association rate approaches the diffusion limit of the protein, vesicle reaction. The association of the protein with lipid vesicles is followed by a slower process occurring at observed rate of 0.031 s(-1), involving insertion into the bilayer and generation of a functional oligomer of diacylglycerol kinase. Protein aggregation competes with vesicle insertion. The urea-denatured protein monomers begin to aggregate as soon as the urea is diluted. This aggregation is faster than the association of the protein with vesicles so that most protein aggregates before it inserts into a vesicle. Increasing the vesicle concentration favours insertion of protein monomers, but at high vesicle concentrations monomers are primarily in separate vesicles and do not associate to form functional oligomers. Irreversible aggregation limits the yield of functional protein, while the data also suggest that lipid vesicles can reverse another aggregation reaction, leading to the recovery of correctly folded protein.
膜蛋白折叠一直缺乏详细的动力学研究，尤其是在变性蛋白插入脂质双层方面。我们对变性的跨膜α螺旋蛋白与脂质囊泡的结合进行了详细的体外动力学研究。研究了尿素中部分变性状态的大肠杆菌二酰基甘油激酶的折叠机制。该蛋白与脂质囊泡结合，形成蛋白-囊泡复合物，其表观结合常数为2×10⁶ M⁻¹ s⁻¹。这种结合速率接近蛋白与囊泡反应的扩散极限。蛋白与脂质囊泡结合后，会发生一个较慢的过程，观察到的速率为0.031 s⁻¹，包括插入双层膜并生成二酰基甘油激酶的功能性寡聚体。蛋白聚集与囊泡插入相互竞争。尿素变性的蛋白单体一旦尿素被稀释就开始聚集。这种聚集比蛋白与囊泡的结合更快，以至于大多数蛋白在插入囊泡之前就聚集了。增加囊泡浓度有利于蛋白单体的插入，但在高囊泡浓度下，单体主要存在于单独的囊泡中，不会结合形成功能性寡聚体。不可逆聚集限制了功能性蛋白的产量，而数据还表明脂质囊泡可以逆转另一种聚集反应，从而导致正确折叠蛋白的恢复。
