The activation energy for insertion of transmembrane α-helices is dependent on membrane composition
The activation energy for insertion of transmembrane α-helices is dependent on membrane composition
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DOI：
10.1016/s0022-2836(02)00342-x
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发表时间：
2002-06-07
影响因子：
5.6
通讯作者：
Booth, PJ
中科院分区：
文献类型：
--
作者：
Meijberg, W;Booth, PJ
对膜蛋白折叠和组装的物理机制知之甚少。看来脂质双层的某些性质影响膜蛋白在体外折叠，无论是通过调节螺旋插入或包装。为了开始理解这种效应的起源，我们使用水溶性的丙氨酸基肽KKAAAAAAAAAAAAAAAAKKK-酰胺研究脂质力对跨膜α-螺旋插入的影响。该肽在中性pH下与预先形成的1，2-二油酰-L-α-磷脂酰胆碱（DOPC）囊泡结合，但仅在高pH下当Lys残基去质子化时，才直接从水相自发跨膜螺旋插入。这些结果表明，电荷的易位是插入的活化能的主要决定因素。在高pH值下的插入过程的时间分辨测量表明具有约30和430秒的时间常数的双相动力学。较慢的阶段似乎与主要跨膜α-螺旋构象的形成相关，这是根据色氨酸残基转移到膜的碳氢化合物区域来确定的。温度依赖性的测量表明，插入只能在一定的阈值温度以上进行，并且Arrhenius活化能为90 kJ mol（-1）的数量级。动力学、阈值温度和活化能随1，2-二油酰-L-α-磷脂酰乙醇胺（DOPE）引入DOPC膜的摩尔分数而变化。活化能随着DOPE含量的增加而增加，这可以反映这样的事实，即这种脂质驱动双层向非双层转变，并增加了脂质链区域中的侧向压力。这表明，折叠事件涉及插入的螺旋片段的双分子层可以控制的脂力。(C)2002爱思唯尔科技有限公司版权所有。
The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane a-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane a-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces. (C) 2002 Elsevier Science Ltd. All rights reserved.