Transport across membranes via electrostatic 'charge zippers'

我们最近已经鉴定出“电荷拉链”作为膜蛋白中的新结构基序[Walther et al.，Cell，2013]。在这样的系统中，带电荷的氨基酸沿着蛋白质序列以准对称的互补模式排列。这表明盐桥的长梯可以在两亲性α-螺旋片段之间形成。通过跨越疏水性脂质双层，这些电荷拉链可以选择性地将质子运输穿过膜或导致更大的离子传导孔的组装。两个肽系统的3D结构和寡聚体架构，我们已经预测了这样的基序，应在这里的特点，以分析假设的电荷拉链的功能相关性。首先，我们集中在细菌肽TisB（29个氨基酸），这是在E。大肠杆菌在胁迫下诱导生物膜的形成。我们的初步功能测定和固态NMR分析表明，TisB插入到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，因此能够选择性地跨膜运输质子（Oder OH-离子）。作为概念的实验证明，我们计划使用固态NMR距离测量和单体的自旋计数来证明这一点，并详细研究如何形成预期的盐桥。与此同时，分子动力学模拟与经验力场和统计热力学计算将被采用。基于通过TisB二聚体运输过程中离子的自由能分布，应该可以区分不同的可能机制。结合量子力学/分子力学的方法将产生一个详细的局部相互作用的描述。在第二部分中，我们将采用已建立的实验和计算方法与TisB进一步研究抗菌肽Dermicdin从人体汗液。长度为48个氨基酸，这种两亲性螺旋序列最初与细菌膜表面结合，如我们使用定向圆二色性所示。根据电荷拉链模型，如果蛋白质折叠成螺旋发夹并组装成寡聚孔，则可以形成多达7个分子内和分子间盐桥。基于空间参数，皮杀菌素将具有跨越脂质双层的最佳长度，并通过高离子电导率杀死细菌。

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.