Grant: Research Tools and Instruments - Category 1 (<$150,000)

The cell surface is the major interaction region between cells their environment. Cells construct barriers to prevent physical and chemical damage and to mediate the flow of nutrients and waste. All cells have a lipid membrane barrier, and in many cases, a cell wall constructed of peptidoglycan (bacteria) or carbohydrate and lignin (plants). Cell surfaces are frequently coated with additional carbohydrate molecules that are either secreted or attached to surface-anchored proteins or lipids. We are investigating the functions of proteins and carbohydrates that inhabit these critical interfaces. Examples include membrane proteins that function to transport nutrients in or materials out of the cell; secreted enzymes that bind to and digest insoluble material at the cell surface; and cell surface sensory receptors that initiate signals to inform cellular regulatory networks. To understand the mechanism of action of these proteins, we need to define which molecules they bind, how well they bind, and at what rates. Several technologies are available to characterise the binding thermodynamics and kinetics of the interactions involving proteins. However, surface plasmon resonance (SPR) has two distinct advantages. First, the method requires that one of the partners to be characterized be immobilized on a surface chip. For our studies, this is ideal as insoluble components found on cell surfaces, such as lipid membranes, and cell wall materials, such as large carbohydrates and peptidoglycan, can be readily immobilized on the sensor chip. The second advantage is that, in many cases, both the association/dissociation kinetics and equilibrium thermodynamic properties of the interaction can be measured simultaneously over a wide range of conditions. Accordingly, we are proposing to purchase a SPR instrument to conduct such binding experiments in a variety of cell surface systems that share a common feature of one insoluble partner.