Probing biological systems at the single molecule level: Studies of the homophilic binding of the neural cell adhesion molecule (NCAM) and phase dependent lipid anchorage strength.

摘要

Weak non-covalent interactions govern many of life's basic functions including biological recognition, signaling, and transport processes. This thesis focuses on the use of single molecule force probe measurements to systematically investigate these types of bonds and their response to force. This work discusses two distinct biological systems: the homophilic binding of the neural cell adhesion molecule (NCAM) and the anchorage strength of a lipid within a membrane.; The force probe technique implemented in these experiments was the atomic force microscope. In this technique, a tip and sample, sparsely coated with a protein of interest, are brought into contact allowing possible bond formation. The tip is then retracted from the surface applying an external force on any protein complex which formed during contact. Application of force lowers the bond's energy barrier and increases the dissociation rate of the bond, thereby allowing for bond rupture. By retracting the tip at various rates we were able to systematically apply force to the bound complex. This allowed investigation of the bond over a spectrum of force revealing important kinetic information.; This technique was used to probe the homophilic binding interaction of NCAM, an adhesion molecule important in the development of tissue in the peripheral and central nervous system. Current proposals describing the NCAM binding mechanism, however, remain contradictory in nature. For this reason both full-length NCAM and deletion mutants were used to study this interaction. Data involving the full-length protein revealed that two separate binding events were involved in the homophilic binding of NCAM. Mutated proteins with various immunoglobulin (Ig) domains deleted were then used to determine the involvement of particular Ig domains in each binding event.; Force probe measurements were also used to study environmental effects on the intermolecular strength of lipids within a membrane. In this case the anchorage strength of lipids in fluid versus gel lipid layers was probed to determine how temperature affects the adhesion properties between adjacent lipid molecules. From this study we have shown that it takes almost three times the force to extract a lipid from a gel phase lipid layer as opposed a fluid phase layer.