Ladokhin Alexey S, White Stephen H
Department of Physiology and Biophysics and Program in Macromolecular Structure, University of California, Irvine, California 92697-4560, USA.
Biochemistry. 2004 May 18;43(19):5782-91. doi: 10.1021/bi0361259.
Nonconstitutive membrane-active proteins, such as diphtheria toxin, must refold on membrane interfaces in the course of membrane penetration. A useful step in deciphering this process is to understand quantitatively the energetics of interface-mediated insertion of model transmembrane helices. A difficulty is that peptides that are sufficiently hydrophobic to span a lipid bilayer have a strong tendency to aggregate in the aqueous phase. To learn how to control the aqueous and membrane behavior of model peptides, we designed a 31-residue peptide (TMX-3) whose properties are described here. TMX-3 has two important structural features: a proline residue in the hydrophobic core that discourages the formation of highly helical aggregates in solution and two histidine residues that allow control of membrane and solution interactions by means of pH changes. The partitioning of TMX-3 into membranes followed complex kinetics, induced helicity, and shifted the histidine pK(a) from 6.8 to approximately 6. Topology measurements disclosed two general modes of TMX-3 binding: interfacial (IF) at low peptide concentrations and partial transmembrane (TM) insertion at higher concentrations. Both modes were reversible and, consequently, suitable for thermodynamic analysis. The free energies of IF partitioning of TMX-3 with deprotonated (pH 7.6) and protonated histidines (pH 4.5) were estimated by fluorescence titration to be -6.7 and -5.0 kcal/mol, respectively. These results show that histidine titration is likely to be important in the pH-dependent refolding of toxins on membrane interfaces and that the most favored state of TMX-3 under any conditions is the IF folded state, which emphasizes the importance of such states in the spontaneous refolding and insertion of diphtheria and other membrane toxins.
非组成型膜活性蛋白，如白喉毒素，在膜穿透过程中必须在膜界面上重新折叠。解读这一过程的一个有用步骤是定量理解模型跨膜螺旋的界面介导插入的能量学。一个困难在于，疏水性足以跨越脂质双层的肽在水相中具有强烈的聚集倾向。为了了解如何控制模型肽在水相和膜中的行为，我们设计了一种31个残基的肽（TMX-3），其性质在此处描述。TMX-3有两个重要的结构特征：疏水核心中的一个脯氨酸残基，可抑制溶液中高度螺旋聚集体的形成；以及两个组氨酸残基，可通过pH变化控制膜与溶液的相互作用。TMX-3在膜中的分配遵循复杂的动力学，诱导螺旋度，并将组氨酸的pK(a)从6.8转变为约6。拓扑测量揭示了TMX-3结合的两种一般模式：低肽浓度下的界面（IF）结合和高浓度下的部分跨膜（TM）插入。两种模式都是可逆的，因此适用于热力学分析。通过荧光滴定估计，去质子化（pH 7.6）和质子化组氨酸（pH 4.5）时TMX-3的IF分配自由能分别为-6.7和-5.0 kcal/mol。这些结果表明，组氨酸滴定可能在毒素在膜界面上的pH依赖性重新折叠中起重要作用，并且在任何条件下TMX-3最有利的状态是IF折叠状态，这强调了这些状态在白喉毒素和其他膜毒素的自发重新折叠和插入中的重要性。
