Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Resolving cadherin interactions and binding cooperativity at the single-molecule level
Figure Lengend Snippet: Close proximity of cadherin molecules in a cis orientation cooperatively increases the probability of trans cadherin binding. ( A ) Schematic of AFM tip and substrate functionalized with cadherin–Fc dimers for single-molecule force measurements. The tip and surface were functionalized with PEG linkers, some of which were decorated with streptavidin molecules. Cadherin–Fc dimers were immobilized on biotinylated protein G attached to the streptavidin molecules on the tip and surface. The flexible PEG linkers enable unhindered interactions between opposing cadherins during tip–substrate encounters. Cadherin–Fc dimers were immobilized on the AFM tip and substrate at a surface density of 34 ± 16 dimers per μm 2 . ( B ) Schematic of AFM tip and substrate functionalized with biotinylated cadherin monomers for single-molecule force measurements. The biotinylated cadherin monomers were immobilized on an AFM tip and substrate via streptavidin-PEG tethers at a surface densities of 65 ± 18 monomers per μm 2 ; thus, the protein surface density for the cadherin monomers and cadherin–Fc dimer constructs are similar. ( C ) A typical force curve showing the unbinding of a single cadherin molecule. The tip and the substrate decorated with cadherins were bought into contact for either 1,100, 340, or 115 ms so that cadherins on the tip and substrate formed an adhesive complex. When the tip was withdrawn from the substrate a force was exerted, and above a critical force the adhesive complex ruptured. Forces between the cadherin–Fc dimers were measured 7,995 times in 2.5 mM Ca 2+ , yielding 509 binding events, and 5,931 times in 2 mM EGTA, yielding 123 binding events. Forces between the cadherin monomers were measured 5,949 times in 2.5 mM Ca 2+ , yielding 113 binding events, and 5,846 times in 2 mM EGTA, yielding 28 binding events. ( D ) Histogram of cadherin–Fc dimer binding events measured in 2.5 mM Ca 2+ (solid gray bars) and 2 mM EGTA (hatched green bars). The binding events measured in 2.5 mM Ca 2+ were fitted to a Gaussian distribution with a peak force of 53 ± 27 pN. The binding probability of cadherin–Fc dimers at contact times of 1,100, 340, or 115 ms was 8.6%, 6.6%, and 3.7%, respectively. The histogram includes the forces measured at all 3 tip-surface contact times. ( E ) Histogram of cadherin monomer binding events measured in 2.5 mM Ca 2+ (solid gray bars) and 2 mM EGTA (hatched green bars). The histogram includes the forces measured at all 3 tip-surface contact times (see above). The binding events measured in 2.5 mM Ca 2+ were fitted to a Gaussian distribution with a peak force of 64 ± 27 pN. Although the bond strengths of the monomer and cadherin–Fc dimer adhesive complexes are similar, the cadherin–Fc dimer has a higher probability of binding than a cadherin monomer. The total probability of cadherin monomer binding at contact times of 1,100, 340, or 115 ms was 2.5%, 2.2%, and 1.1% respectively. Thus, the cadherin–Fc dimer binding probability was 3.5, 3.1, and 3.6 times higher than the probability of monomer interactions at these tip-surface contact times.
Article Snippet: To immobilize Fc–cadherin dimers, the streptavidins were decorated with biotinylated protein G (Pierce Biotechnology), and the Fc region of the cadherin dimer was specifically bound to the protein G. Fluorescently-labeled cadherin molecules were observed by using a home-built prism-type total internal reflection fluorescence microscope.
Techniques: Binding Assay, Construct, Mass Spectrometry