Multiple-bond kinetics from single-molecule pulling experiments: Evidence for multiple NCAM bonds

The kinetic parameters of single bonds between neural cell adhesion molecules were determined from atomic force microscope measurements of the forced dissociation of the homophilic protein-protein bonds. The analytical approach described provides a systematic procedure for obtaining rupture kinetics for single protein bonds from bond breakage frequency distributions obtained from single-molecule pulling experiments. For these studies, we used the neural cell adhesion molecule (NCAM), which was recently shown to form two independent protein bonds. The analysis of the bond rupture data at different loading rates, using the single-bond full microscopic model, indicates that the breakage frequency distribution is most sensitive to the distance to the transition state and least sensitive to the molecular spring constant. The analysis of bond failure data, however, motivates the use of a double-bond microscopic model that requires an additional kinetic parameter. This double-bond microscopic model assumes two independent NCAM-NCAM bonds, and more accurately describes the breakage frequency distribution, particularly at high loading rates. This finding agrees with recent surface-force measurements, which showed that NCAM forms two spatially distinct bonds between opposed proteins.