Transport across membranes via electrostatic 'charge zippers'

我们最近发现“电荷拉链”是膜蛋白中的一种新型结构基序[Walther et al., Cell, 2013]。在这种系统中，带电荷的氨基酸沿蛋白质序列以准对称的互补模式排列。这表明盐桥的长阶梯可以在两亲性α -螺旋段之间形成。通过跨越疏水脂质双分子层，这些电荷拉链可以选择性地将质子传输到膜上，或者导致更大的离子传导孔的组装。为了分析假设的电荷拉链的功能相关性，我们将对我们预测的两个肽系统的三维结构和低聚结构进行表征。首先，我们关注细菌肽TisB（29个氨基酸），它在大肠杆菌中产生并诱导生物膜的形成。我们的初步功能分析和固态核磁共振分析表明，将TisB插入膜中导致质子梯度的受控耗尽。首先，粗粒度和全原子模拟支持我们的模型，根据该模型，TisB通过4个盐桥组装成反平行二聚体，因此能够选择性地在膜上传输质子（较旧的OH-离子）。作为概念的实验证明，我们计划使用固态核磁共振距离测量和单体的自旋计数来证明这一点，并详细研究预期的盐桥是如何形成的。同时，分子动力学模拟与经验力场和统计热力学计算将被采用。基于离子在通过TisB二聚体传输过程中的自由能分布，应该可以区分不同的可能机制。结合量子力学/分子力学方法将产生局部相互作用的详细描述。在第二部分，我们将利用TisB已经建立的实验和计算方法进一步研究人体汗液中的抗菌肽Dermicdin。长度为48个氨基酸，这个两亲性螺旋序列最初与细菌膜表面结合，正如我们使用定向圆二色性所示。根据电荷拉链模型，如果蛋白质像螺旋发夹一样折叠并组装成一个低聚孔，那么可以形成多达7个分子内和分子间的盐桥。基于立体理论，Dermcidin将具有跨越脂质双分子层的最佳长度，并通过高离子导电性杀死细菌。

We have recently identified 'charge zippers' as a novel structural motif in membrane proteins [Walther et al., Cell, 2013]. In such systems the charged amino acids are aligned in a quasi-symmetrical, complementary pattern along the protein sequence. This suggests that long ladders of salt bridges can be formed between amphiphilic alpha-helical segments. By spanning the hydrophobic lipid bilayer, these charge zippers could selectively transport protons across membranes or lead to the assembly of larger ion-conducting pores. The 3D structures and oligomeric architectures of two peptide systems, for which we have predicted such motif, shall be characterized here, in order to analyze the functional relevance of the postulated charge zippers. First, we focus on the bacterial peptide TisB (29 amino acids), which is produced in E. coli under stress and induces the formation of biofilms. Our preliminary functional assays and solid-state NMR analysis have shown that the insertion of TisB into a membrane leads to a controlled depletion of the proton gradient. First coarse-grained and all-atom simulations support our model, according to which TisB assembles into antiparallel dimers via 4 salt bridges and is thus able to selectively transport protons (oder OH- ions) across the membrane. As an experimental proof of concept we plan to use solid-state NMR distance measurements and spin-counting of the monomers to demonstrate that and study in detail how the expected salt bridges are formed. In parallel, molecular dynamics simulations with empirical force fields and calculations in statistical thermodynamics will be employed. Based on the free energy profiles of the ions during transport via a TisB dimer it should be possible to discriminate the different possible mechanisms. Combined quantum mechanical/molecular mechanical methods shall yield a detailed description of the local interactions.In the second part, we will employ the experimental and computational methods that have been established with TisB further to study the antimicrobial peptide Dermicdin from human sweat. With a length of 48 amino acids, this amphiphilic helical sequence binds initially to the surface of bacterial membranes, as we have shown using oriented circular dichroism. According to the charge zipper model, up to 7 intra- and intermolecular salt bridges could then be formed if the protein folds as a helical hairpin and assembles into an oligomeric pore. Based on sterical arguments, Dermcidin would then possess the optimal length to span a lipid bilayer and kill the bacteria by high ion conductivity.