| This thesis utilizes single molecule techniques of atomic force microscopy and polymer physics to study force-driven conformational changes and extensibility of various proteins. The proteins are considered model polymers with complex structures. Their flexibility as well as the submolecular interactions within and between protein molecules are probed in competition with surface adsorption interactions. The proteins studied are various constructs of triple-helix family of spectrin proteins, which are prototypical in being extensible, multi-domain proteins. Spectrin, Dystrophin, and alpha-Actinin were all studied and widely recognized for their various contributions to erythrocyte and muscle flexibility. Recent atomic level data not only confirms coiled triple helical repeating units but also suggests that the linker between each spectrin repeat is a contiguous helix. This raises questions as to what the linker contributes to stability and what defines an independent folded domain mechanically. This thesis examined the extensible unfolding of nearly a dozen spectrin family proteins as monomeric constructs of two, three, four, five, and eight repeats or mixed as dimeric constructs. An ultimate aim is to relate extensible unfolding of spectrins to the mechanical resilience of cell membranes and muscle. In this context, the dystrophin peptides studied included an eight repeat construct and a five repeat engineered construct used in gene therapy (on mice with muscular dystrophy) but of unknown mechanical properties. The latter construct possesses an interesting proline-rich hinge linker. In addition, thermal effects in unfolding these proteins as well as the thermodynamic free energy changes induced by temperature during the unfolding process were explored. |
