van den Elsen J M, van Unen L M, van Bloois L, Busquets M A, Jiskoot W, Hoogerhout P, Wilting J, Herron J N, Crommelin D J
Department of Pharmaceutics, Faculty of Pharmacy, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands.
Biochemistry. 1997 Oct 14;36(41):12583-91. doi: 10.1021/bi9700431.
An antibody-peptide model system was used to study the binding characteristics between a bactericidal antibody (MN12H2) and the P1. 16 epitope of class 1 outer membrane protein PorA of Neisseria meningitidis by means of a thermodynamic approach. A series of four linear peptides and three "head-to-tail" cyclic peptides (with ring sizes of 9, 15 and 17 amino acids) were synthesized and evaluated as ligands. The peptides contain a fluorescein label and the core determinant amino acid sequence TKDTNNN (residues 180-186) of the PorA P1.16 epitope of meningococcal strain H44/76. Thermodynamic data of the binding of the peptide homologs of the epitope by MN12H2 were assessed by measuring affinity constants (Ka) over a temperature range of 4-55 degrees C, using fluorescence spectroscopy. Curvilinear plots of ln Ka versus T (K) revealed strong temperature dependencies of enthalpy (DeltaH) and entropy (DeltaS). The Gibbs free energy change (DeltaG) was only weakly temperature dependent. The large negative enthalpy value indicated the importance of polar interactions in the binding of both linear and cyclic peptides by MN12H2. Sturtevant's analysis of the thermodynamic parameters showed large unfavorable vibrational contributions to the binding for all linear peptides [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci.U.S.A. 74, 2236-2240]. The large hydrophobic contribution compensating these vibrational modes was partially attributed to aspecific interaction of the fluorescein label with the antibody. Binding of MN12H2 to conformationally restricted epitope sequences was characterized by a dramatic reduction in the size of unfavorable vibrational components of the thermodynamic parameters. Substitution of individual charged amino acids of the P1.16 epitope sequence revealed that aspartate-182 was essential for the binding. The pH profile observed for the MN12H2-peptide complexes with a midpoint pH of approximately 8.5 suggests a positively charged histidine from the antibody binding site to be involved in a charge interaction with Asp-182. These findings are consistent with the results from the crystal structure of the Fab fragment of MN12H2 in complex with a linear fluorescein-conjugated peptide homolog of the P1.16 epitope [van den Elsen et al. (1997) Proteins (in press)], thereby identifying the basis of an increased incidence of endemic disease in England and Wales since 1981 caused by a mutant meningococcal strain.
利用抗体 - 肽模型系统，通过热力学方法研究杀菌抗体（MN12H2）与脑膜炎奈瑟菌1类外膜蛋白PorA的P1.16表位之间的结合特性。合成了一系列四种线性肽和三种“头对尾”环肽（环大小分别为9、15和17个氨基酸），并将其作为配体进行评估。这些肽含有荧光素标记以及脑膜炎球菌菌株H44/76的PorA P1.16表位的核心决定氨基酸序列TKDTNNN（第180 - 186位残基）。通过荧光光谱法在4 - 55℃温度范围内测量亲和常数（Ka），评估MN12H2与该表位肽类似物结合的热力学数据。ln Ka对T（K）的曲线表明焓（ΔH）和熵（ΔS）强烈依赖于温度。吉布斯自由能变化（ΔG）仅对温度有微弱依赖。大的负焓值表明极性相互作用在MN12H2与线性和环肽结合中具有重要性。对热力学参数的斯特蒂文特分析表明，所有线性肽的结合都有很大的不利振动贡献[斯特蒂文特，J.M.（1977年）美国国家科学院院刊74，2236 - 2240]。补偿这些振动模式的大的疏水贡献部分归因于荧光素标记与抗体的非特异性相互作用。MN12H2与构象受限表位序列的结合特征是热力学参数中不利振动成分的大小显著减小。对P1.16表位序列中单个带电氨基酸的取代表明，天冬氨酸 - 182对结合至关重要。观察到MN12H2 - 肽复合物的pH曲线中点pH约为8.5，这表明来自抗体结合位点的带正电组氨酸参与了与天冬氨酸 - 182的电荷相互作用。这些发现与MN12H2的Fab片段与P1.16表位的线性荧光素缀合肽类似物复合物的晶体结构结果一致[范登·埃尔森等人（1997年）蛋白质（即将发表）]，从而确定了自1981年以来由突变脑膜炎球菌菌株导致的英格兰和威尔士地方病发病率增加的基础。
