EXCEED THE SPACE PROVIDED. Diabetes is a chronic disease that is approaching epidemic proportions with the ageing of the US population. At present there is no established cure, but patients can lead near-normal lives with a healthy lifestyle, new and more effective drugs and constant blood glucose measurements. However, available glucose monitoring systems are inconvenient, costly and can be done only a few times during the day. Recent continuous glucose monitors approved by the FDA have limited use and suffer from drift due to the degradation of the glucose oxidase that is the heart of the sensing mechanism. Several groups, including our own, have investigated other glucose binding proteins as alternative to glucose oxidase with some degree of success. This proposal seeks to go beyond single proteins as glucose sensors, rather we will look at glucose-induced protein-protein interactions as they occur in nature. This approach will utilize the glucose permease system of E. coli, an ABC transporter comprised of the periplasmic glucose binding protein (GBP or MglB), and two copies each of a membrane spanning protein (MglC) and an ATPase (MglA). Specifically, we will: 1. Overexpress and isolate these proteins in E. coli by recombinant techniques. 2. Label GBP and MglC with appropriate FRET donor/acceptor pairs. 3. Reconstitute MglC and MglA in polymeric liposomes or membranes. 4. Evaluate glucose response by measuring FRET in MgIB/MglC/MglA (micromolar glucose sensitivity) and MglC/MglA (millimolar glucose sensitivity). 5. Devise a reagentless glucose sensor by designing MglA mutants that lack ATPase activity but respond to glucose binding. The long-term goal is to develop a low-cost optical glucose sensor for in vivo monitoring. Instrumentation, chemical and immobilization technologies are already in place. All that is needed is a signal transducer that is specific for glucose, has the right sensitivity and is reliable for long periods. This proposal seeks funding for the development of a new type of biosensor based on the ABC transporter system that may pave the way for a broader range of biomedically important analytes. PERFORMANCE SITE ========================================Section End===========================================
