Thermodynamic analysis of the interaction between a bactericidal antibody and a PorA epitope of Neisseria meningitidis

Publication Type:

Journal Article


Biochemistry, Volume 36, Number 41, pp. 12583-12591 (1997)




outer-membrane protein; molar heat-capacity; amino-acid-residues; synthetic peptides; fluorescence polarization; binding thermodynamics; antigen interactions; monoclonal-antibody; sequences; lysozyme


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 (K-a) over a temperature range of 4-55 degrees C, using fluorescence spectroscopy. Curvilinear plots of In K-a versus T (K) revealed strong temperature dependencies of enthalpy (Delta H) and entropy (Delta S). The Gibbs free energy change (Delta G) 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.