Constantinescu Iren, Lafleur Michel
Department of Chemistry, Université de Montréal, CP 6128, Succ. Centre Ville, Montréal, Québec, Canada, H3C 3J7.
Biochim Biophys Acta. 2004 Nov 17;1667(1):26-37. doi: 10.1016/j.bbamem.2004.08.012.
We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the 19Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the 19Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an alpha-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of 19Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the alpha-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.
我们使用三种独立且互补的方法，研究了从蜂毒中提取的二级两亲性肽蜂毒肽在脂质膜上的吸附动力学。我们探究了：（i）肽结合后19位色氨酸极性的变化；（ii）该残基插入膜的非极性核心，通过连接在脂质酰基链上的溴原子测量19位色氨酸荧光猝灭；以及（iii）通过圆二色性（CD）研究肽的折叠。我们报道了肽的插入与其折叠成α-螺旋之间紧密的耦合关系。对于所有研究的膜系统（含胆固醇的、含磷酸甘油的和纯磷脂酰胆碱双层膜），发现19位色氨酸极性的降低明显快于蜂毒肽螺旋含量的增加。因此，从动力学角度来看，α-螺旋的形成是蜂毒肽插入的结果。发现蜂毒肽的折叠速率受双层膜脂质组成的影响，我们认为这是通过调节插入动力学实现的。该研究报道了一个蛋白质渗透与折叠之间存在耦合关系的清晰例子，在膜蛋白折叠的总体框架中必须考虑这种相互联系。
