Peptide backbone chemistry and membrane channel function: effects of a single amide-to-ester replacement on gramicidin channel structure and function.
Peptide backbone chemistry and membrane channel function: effects of a single amide-to-ester replacement on gramicidin channel structure and function.
复制标题
肽主链化学和膜通道功能：单一酰胺到酯的取代对短杆菌肽通道结构和功能的影响。
DOI：
10.1021/bi001562y
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Koeppe2nd,R
中科院分区：
文献类型：
--
作者：
Jude,AR;Providence,LL;Schmutzer,SE;Shobana,S;Greathouse,DV;Andersen,OS;Koeppe2nd,R
为了研究离子通道中主干酰胺基团在亚基折叠、氢键、离子溶剂化和离子渗透方面的结构和功能重要性，我们用酯键取代了val1和Gly2in gramicidin A之间的肽键。取代发生在两个通道亚基之间的连接处，它消除了gly2的NH和val7的CO之间的分子内氢键，并扰乱了val1的CO和ala5的NH在另一个亚基中的分子间氢键。因此，取代不仅扰乱了亚基折叠，而且扰乱了二聚体组装，以及对离子渗透的任何影响。这种主链修饰对通道功能有很大的影响：它改变了通道稳定性（通过通道形成能力和通道寿命来监测）和离子渗透率（通过单通道电导和阳离子渗透率比的变化来监测）。事实上，具有两个含酯亚基的同二聚体通道的寿命非常短，以至于不可能对其进行任何详细的表征。然而，肽→酯取代不影响基本亚基折叠，因为异二聚体通道可以在具有酯键的亚基和天然亚基之间形成。这些异二聚体通道，只有一个单一的酯键，更容易表征；与天然的同二聚体通道相比，单酯将单通道电导降低约4倍，寿命降低约200倍。通道功能的改变是由于氢键网络的扰动/破坏造成的，氢键网络稳定了骨架和跨膜二聚体，并形成了离子传导孔的内衬。分子动力学模拟表明，修饰后的异二聚体或同二聚体通道具有预期的不稳定性，但主链结构和动力学变化非常小。酯键有些不稳定，这妨碍了进一步的结构表征。这种不稳定性也导致水解产物以醇终止，缺乏甲酰基戊。水解产物形成的对称通道寿命也很短，但与革兰杀菌酯a形成的对称通道明显不同。此外，在水解产物和参考亚基之间形成的不对称通道表现良好，这些参考亚基在甲酰基- n -端有反基或反基残基。
To examine the structural and functional importance of backbone amide groups in ion channels for subunit folding, hydrogen bonding, ion solvation, and ion permeation, we replaced the peptide bond between Val1and Gly2in gramicidin A by an ester bond. The substitution is at the junction between the two channel subunits, where it removes an intramolecular hydrogen bond between the NH of Gly2and the CO of Val7and perturbs an intermolecular hydrogen bond between the CO of Val1in one subunit and the NH of Ala5in the other subunit. The substitution thus perturbs not only subunit folding but also dimer assembly, in addition to any effects on ion permeation. This backbone modification has large effects on channel function: It alters channel stability, as monitored by the channel forming ability and channel lifetime, and ion permeability, as monitored by changes in single-channel conductance and cation permeability ratios. In fact, the homodimeric channels, with two ester-containing subunits, have lifetimes so short that it becomes impossible to characterize them in any detail. The peptide → ester substitution, however, does not affect the basic subunit fold because heterodimeric channels can form between a subunit with an ester bond and a native subunit. These heterodimeric channels, with only a single ester bond, are more easily characterized; the lone ester reduces the single-channel conductance about 4-fold and the lifetime about 200-fold as compared to the native homodimeric channels. The altered channel function results from a perturbation/disruption of the hydrogen bond network that stabilizes the backbone, as well as the membrane-spanning dimer, and that forms the lining of the ion-conducting pore. Molecular dynamics simulations show the expected destabilization of the modified heterodimeric or homodimeric channels, but the changes in backbone structure and dynamics are remarkably small. The ester bond is somewhat unstable, which precluded further structural characterization. The lability also led to a hydrolysis product that terminates with an alcohol and lacks formyl-Val. Symmetric channels formed by the hydrolyzed product again have short lifetimes, but the channels are distinctly different from those formed by the ester gramicidin A. Furthermore, well-behaved asymmetric channels form between the hydrolysis product and reference subunits that have either anl- or ad-residue at the formyl-NH-terminus.