ASSOCIATION OF PEPTIDES IN MEMBRANES

膜中肽的缔合

• 批准号: 7367745
• 负责人: ANDREW POHORILLE
• 金额: $ 0.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367745
• 关键词: ASSOCIATION; PEPTIDES; MEMBRANES

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Single transmembrane helices are rarely capable of performing biological functions. Instead, they form functional units after self-assembling into higher order structures. However, not all helices self-assemble. Consequently, it is necessary to understand sequence-specific interhelical recognition before we can predict the kinds of structures that can form in membranes. The simplest models for peptide association are helical dimers. Simple experimental models have recently been developed for helix associations in membranes. The best-studied system, both structurally and thermodynamically, is the 24-residue transmembrane region of glycophorin A (GpA). GpA forms non-covalent dimers through the reversible association of its membrane-spanning domain, which adopts an alpha-helical conformation. The structure has recently been determined by NMR spectroscopy of 40-residue peptides that contain the transmembrane segment, solvated in detergent micelles. This study also showed that the alpha-helices formed a right-handed, coil-coiled structure. We simulated the wild-type dimer of GpA in a lamella of dodecane, placed between two lamellae of water. The width of the dodecane layer was approximately the same as that of the hydrophobic core of a palmitoyloleylphosphatidylcholine (POPC) membrane. The starting structure was based on the NMR model of the dimer. The comparison between the calculated, time-averaged structure of the dimer after 30.0 ns of molecular dynamics trajectory and the nuclear Overhauser effect data of MacKenzie, et al., provided an assessment of the accuracy of our model and the potential energy functions utilized. The distance root mean square deviation was less than 1.5 Angstroms for the backbone atoms and 2.75 Angstroms for all atoms (Hydrogens excluded) and the monomers remained alpha-helical. Engelman, et. al, have observed interhelical close contacts between several Hydrogens bound to alpha Carbons and carbonyl Oxygens and argue that these can form weak hydrogen bonds that stablize the dimer. We observe that between 4 and 5 of these contacts are maintained over the course of the simulations and could contribute significantly to the stability of the dimer. The dissociation free energy of the GpA dimer in a detergent pentaoxyethylene (C8E5) has been estimated to be about 9.0 kcal/mol. It has been also demonstrated that single-residue mutations can markedly influence the free energy of association of the helices. Mutants in which either one of two leucine residues were substituted with alanine or glycine was substituted with isoleucine were found to be less stable than the wild-type dimer by 1-3 kcal/mol. All these residues are involved in interhelical interactions in the NMR determined model of GpA. We separated the helices in a series of molecular dynamics simulations using the distance separating the centers of mass of the two helices as the reaction coordinate. The free energy of dissociation was calculated using the Adaptive Biasing Force (ABF) method of Darve and Pohorille. The complete free energy pathway joining the dimer from 5.5 Angstroms at contact to dissociated monomers at 20 Angstroms was divided into a series of intermediate states corresponding to different values of the reaction coordinate. The free energy of dissociation is given as the sum of free energy differences between consecutive intermediate states. The computed free energy is approximately 10.0 kcal/mol. By comparison, the experimentally determined free energy of dissociation in the detergent pentaoxyethylene (C8E5) is equal to 9.0 kcal/mol. Since the molecular environments of GpA in the computational and experimental studies are different - ie. dodecane versus C8E5 - the free energies are not expected to be identical, but should be similar. Given that the volume accessible to the alpha-helical dimer is much smaller in C8E5 than in dodecane, which corresponds to a smaller entropic contribution, we expect that the free energy of dissociation should be higher in dodecane than in C8E5. Based on similar considerations, it is expected that the influence of the surroundings on the computed point mutations would, in principle, be limited due to compensation of entropic effects in the free energy differences. However, this may not be necessarily the case. The I76L point mutation was carried out computationally by decoupling the annihilation of the electrostatic and the van der Waals and internal parameter contributions. The free energy difference for the electrostatic term was 0 kcal/mol to within the statistical errors. The contribution due to the modification of the van der Waals parameters and the participating chemical bonds and valence angles, and, therefore, the total free energy difference, is equal to 0.4 kcal/mol, which is somewhat less than the experimental value of 1.7 kcal/mol. It should be emphasized, however, that, as the L-isoleucine is transformed into L-alanine, the disruptive effect of the mutation found experimentally in C8E5 should be less pronounced in dodecane, because of the greater volume accessible to the alpha-helices in that environment. The free energy change upon single-point mutation is fairly small. In contrast, the free energy of dissociation is large and positive. This might suggest that the dimer is strongly favored for both the wild-type and the mutants. This would indeed be the case if we only considered the equilibrium between the associated state and separated, transmembrane helices. However, this comparison is not appropriate because the transmembrane orientation of helices is not necessarily stable. The free energy of insertion of a helical peptide into the membrane is positive (unfavorable) and may be substantial. Thus, the equilibrium that needs to be considered is between the transmembrane dimer and the individual helices at the water-membrane interface. This equilibrium is governed by the balance between the unfavorable free energy of insertion into the membrane and favorable free energy of interhelical association. This balance could be subtle, and modest changes in either term could shift the equilibrium, possibly disrupting dimerization.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。单跨膜螺旋很少能够发挥生物学功能。相反，它们在自组装成更高阶结构后形成功能单元。然而，并非所有螺旋都能自组装。因此，在我们预测膜中可以形成的结构类型之前，有必要了解序列特异性螺旋间识别。最简单的肽缔合模型是螺旋二聚体。最近开发了用于膜中螺旋缔合的简单实验模型。从结构和热力学角度来说，研究得最好的系统是血型糖蛋白 A (GpA) 的 24 个残基跨膜区域。 GpA 通过其跨膜结构域的可逆缔合形成非共价二聚体，该二聚体采用 α 螺旋构象。最近通过 40 个残基肽的 NMR 光谱确定了该结构，这些肽包含溶剂化在洗涤剂胶束中的跨膜片段。这项研究还表明，α-螺旋形成了右手螺旋结构。我们在放置在两个水层之间的十二烷层中模拟了野生型 GpA 二聚体。十二烷层的宽度与棕榈酰油酰磷脂酰胆碱（POPC）膜的疏水核心的宽度大致相同。起始结构基于二聚体的 NMR 模型。分子动力学轨迹 30.0 ns 后计算出的二聚体时间平均结构与 MacKenzie 等人的核奥弗豪瑟效应数据之间的比较，为我们的模型和所利用的势能函数的准确性提供了评估。主链原子的距离均方根偏差小于 1.5 埃，所有原子（氢除外）的距离均方根偏差小于 2.75 埃，并且单体仍保持 α 螺旋。恩格曼等。等人观察到与α碳和羰基氧结合的几个氢之间的螺旋间紧密接触，并认为这些可以形成稳定二聚体的弱氢键。我们观察到，在模拟过程中，其中 4 到 5 个接触得以维持，并且可能对二聚体的稳定性做出重大贡献。 洗涤剂五氧乙烯 (C8E5) 中 GpA 二聚体的解离自由能估计约为 9.0 kcal/mol。还证明单残基突变可以显着影响螺旋的缔合自由能。发现两个亮氨酸残基之一被丙氨酸取代或甘氨酸被异亮氨酸取代的突变体比野生型二聚体稳定性低1-3 kcal/mol。所有这些残基都参与 NMR 确定的 GpA 模型中的螺旋间相互作用。我们使用两个螺旋质心之间的距离作为反应坐标，在一系列分子动力学模拟中分离螺旋。使用 Darve 和 Pohorille 的自适应偏置力 (ABF) 方法计算解离自由能。连接二聚体（从 5.5 埃接触处到 20 埃处解离单体）的完整自由能路径被分为一系列对应于不同反应坐标值的中间状态。解离自由能作为连续中间态之间的自由能差之和给出。计算出的自由能约为 10.0 kcal/mol。相比之下，实验测定的洗涤剂五氧乙烯 (C8E5) 中的解离自由能等于 9.0 kcal/mol。由于计算和实验研究中 GpA 的分子环境不同 - 即。十二烷与 C8E5 - 自由能预计不会相同，但应该相似。鉴于 C8E5 中 α-螺旋二聚体可到达的体积比十二烷小得多，这对应于较小的熵贡献，我们预计十二烷中的解离自由能应高于 C8E5。基于类似的考虑，由于自由能差异中熵效应的补偿，预计周围环境对计算的点突变的影响原则上将受到限制。然而，情况可能不一定如此。 I76L 点突变是通过解耦静电和范德华的湮灭以及内部参数贡献来计算的。静电项的自由能差异为 0 kcal/mol，在统计误差范围内。由于范德华参数以及参与的化学键和价角的修改而产生的贡献，因此总自由能差等于 0.4 kcal/mol，略小于实验值 1.7 kcal /摩尔。然而，应该强调的是，当 L-异亮氨酸转化为 L-丙氨酸时，在 C8E5 中实验发现的突变的破坏作用在十二烷中应该不那么明显，因为十二烷中的 α-螺旋可接近的体积更大。那个环境。 单点突变时的自由能变化相当小。相反，解离自由能大且正。这可能表明二聚体对于野生型和突变体都非常有利。如果我们只考虑关联态和分离的跨膜螺旋之间的平衡，情况确实会如此。然而，这种比较并不合适，因为螺旋的跨膜方向不一定稳定。将螺旋肽插入膜的自由能是正的（不利的）并且可能很大。因此，需要考虑跨膜二聚体和水膜界面处的各个螺旋之间的平衡。这种平衡由不利的插入膜自由能和有利的螺旋间缔合自由能之间的平衡决定。这种平衡可能是微妙的，任何一项的适度变化都可能改变平衡，可能破坏二聚化。