Single protein intermolecular binding force detection using microfabricated cantilevers

The atomic force microscope (AFM) is an instrument that allows for the measurement of extremely small forces with exceptional accuracy and resolution not only in force, but also in time and location. These are precisely the variables needed for applying thermodynamic theory on a very small scale. In this work, single intermolecular bonds between E-cadherin molecules were distinguished and their strength measured using modified AFM cantilevers. The role of the AFM was extended to incorporate emerging molecular kinetic theory to a single bond and to small numbers of multiple bonds for measurements using the E-cadherin/E-cadherin homotypic bond as a model system.; E-cadherin is a member of the cadherin family of cellular adhesion transmembrane glycoproteins, of which approximately 40 varieties have been identified. Cellular adhesion mediated by these proteins exhibits a strong calcium dependence. In this work the role of calcium in promoting adhesion at a single molecule level is explored by assaying the probability of adhesion and the unbinding force in order to determine if the presence of Calcium affects individually bound pair or whether it works to strengthen or stabilize only within the context of whole-cell mechanics. Fundamental kinetic constants were measured using force spectroscopy. Further, it was shown that calcium has a role in promoting the probability of E-cadherin adhesion, but does not exhibit a functional influence on the nature of the E-cadherin bond.; Within juxtaposed and bound cell membranes, Cadherins have been shown to coalescence into high density immobile aggregates, called puncta. Although these aggregates have been observed to associate with actin filaments on the C-terminal (cytoplasmic) side within the cell, it is not known whether these high density regions further strengthen cellular adhesion through higher order associations in the extracellular domain.; In order to determine the whether such cooperativity is a factor in E-cadherin adhesion, the role of molecular density was explored through measurements done by varying the density and numbers of molecules interacting. Extending kinetic theory to the resulting force density spectra, it was determined that cooperativity in the extracellular domain increases the strength of E-cadherin trans-adhesion and can be easily observed in small numbers of molecules (e.g. 1 < N < 8).