Wieprecht T, Beyermann M, Seelig J
Department of Biophysical Chemistry, Biocenter of the University of Basel, Switzerland.
Biochemistry. 1999 Aug 10;38(32):10377-87. doi: 10.1021/bi990913+.
Magainins are positively charged amphiphatic peptides which permeabilize cell membranes and display antimicrobial activity. They are usually thought to bind specifically to anionic lipids, and binding studies have been performed almost exclusively with negatively charged membranes. Here we demonstrate that binding of magainins to neutral membranes, a reaction which is difficult to assess with spectroscopic means, can be followed with high accuracy using isothermal titration calorimetry. The binding mechanism can be described by a surface partition equilibrium after correcting for electrostatic repulsion by means of the Gouy-Chapman theory. Unusual thermodynamic parameters are observed for the binding process. (i) The three magainin analogues that were investigated bind to neutral membranes with large exothermic reaction enthalpies DeltaH of -15 to -18 kcal/mol (at 30 degrees C). (ii) The reaction enthalpies increase with increasing temperature, leading to a large positive heat capacity DeltaC(p) of approximately 130 cal mol(-)(1) K(-)(1) (at 25 degrees C). (iii) The Gibbs free energies of binding DeltaG are between -6.4 and -8.6 kcal/mol, resulting in a large negative binding entropy DeltaS. The binding of magainin to small unilamellar vesicles is hence an enthalpy-driven reaction. The negative DeltaH and DeltaS and the large positive DeltaC(p) contradict the conventional understanding of the hydrophobic effect. CD experiments reveal that the membrane-bound fraction of magainin is approximately 80% helical at 8 degrees C, decreasing to approximately 60% at 45 degrees C. Since the random coil --> alpha-helix transition in aqueous solution is known to be an exothermic process, the same process occurring at the membrane surface is shown to account for up to 65% of the measured reaction enthalpy. In addition to membrane-facilitated helix formation, the second main driving force for membrane binding is the insertion of the nonpolar amino acid side chains into the lipid bilayer. It also contributes a negative DeltaH and follows the pattern for the nonclassical hydrophobic effect. Addition of cholesterol drastically reduces the extent of peptide binding and reveals an enthalpy-entropy compensation mechanism. Membrane permeability was measured with a dye assay and correlated with the extent of peptide binding. The level of dye efflux is linearly related to the amount of surface-bound peptide and can be traced back to a membrane perturbation effect.
蛙皮素是带正电荷的两亲性肽，可使细胞膜通透并具有抗菌活性。通常认为它们与阴离子脂质特异性结合，并且几乎所有的结合研究都是在带负电荷的膜上进行的。在这里，我们证明了蛙皮素与中性膜的结合（这一反应难以用光谱手段评估）可以通过等温滴定量热法高精度地跟踪。通过古依-查普曼理论校正静电排斥后，结合机制可用表面分配平衡来描述。观察到结合过程具有异常的热力学参数。（i）所研究的三种蛙皮素类似物与中性膜结合时具有较大的放热反应焓ΔH，为 -15至 -18千卡/摩尔（在30℃时）。（ii）反应焓随温度升高而增加，导致在25℃时具有约130卡摩尔⁻¹K⁻¹的大正热容ΔC(p)。（iii）结合的吉布斯自由能ΔG在 -6.4至 -8.6千卡/摩尔之间，导致大的负结合熵ΔS。因此，蛙皮素与小单层囊泡的结合是一个焓驱动的反应。负的ΔH和ΔS以及大的正ΔC(p)与对疏水效应的传统理解相矛盾。圆二色性实验表明，在8℃时，与膜结合的蛙皮素部分约80%呈螺旋状，在45℃时降至约60%。由于已知水溶液中无规卷曲→α-螺旋转变是一个放热过程，在膜表面发生的相同过程被证明占测量反应焓的高达65%。除了膜促进的螺旋形成外，膜结合的第二个主要驱动力是非极性氨基酸侧链插入脂质双层。它也贡献一个负的ΔH，并遵循非经典疏水效应的模式。添加胆固醇会显著降低肽结合的程度，并揭示出一种焓-熵补偿机制。用染料测定法测量膜通透性，并与肽结合程度相关联。染料外排水平与表面结合肽的量呈线性相关，并且可以追溯到膜扰动效应。
