In most living organisms, including animals, plants and bacteria, glucose is employed as energy source, for energy storage and in the synthesis of essential biological molecules. Glucose measurements are critical to elucidate the basic characteristics of glucose distribution at the cellular and subcellular level under normal conditions and in disease states. This is particularly important because in diabetes mellitus, one of the most common chronic diseases in humans, alterations in glucose metabolism (not well understood at the cell level) are the most important factor. Currently, there is no glucose sensor suitable for measuring glucose in cells and the sensors available for clinical and industrial applications are frequently unsatisfactory. This proposal presents a novel approach to develop glucose sensors based on the use of autofluorescent proteins (proteins that fluoresce in the absence of cofactors) and the glucose binding properties of the enzyme glucokinase (hexokinase IV), the glucose sensor protein of the pancreas. The most useful autofluorescent proteins derive from the green fluorescent protein from the jellyfish Aequoria victoria and the red fluorescent protein from a coral of the Discosoma genus (red fluorescent protein). Our plan is to develop a sensor that will be: 1) Suitable for """"""""in vitro"""""""" measurements of glucose concentration, and 2) Genetically-encoded for targeting to specific cells and subcellular compartments, as well as specific cells and tissues of transgenic animals. The sensor can also be evaluated as the backbone for a continuous minimally invasive glucose monitoring system in diabetic patients. We plan to engineer glucose sensors by fusion of glucokinase to autofluorescent proteins. We will purify the sensor proteins expressed in E. coil, analyze their properties (fluorescence properties, response to glucose, specificity) and test their usefulness for measuring glucose in living cells. Although the goal of the project is to obtain a sensor for measurements in cells, to foster basic research under normal and disease conditions, potentially the new sensors can be adapted to industrial (e.g., glucose measurements in fruit juices) and medical (monitoring glycemia in diabetes mellitus) uses.
