Recognition of carbohydrates is one of the important biological signals. It is important in fertilization, cell development, intracellular trafficking of proteins, and other important biological functions. In health-related areas, viral infection requires recognition of certain carbohydrates (e.g., influenza), and adhesion of lymphocytes (an initial step of inflammation) requires recognition of specific carbohydrates. Understanding of recognition of carbohydrates requires thorough knowledge of conformational structure, because branched oligosaccharides found in nature can assume many different conformations only one (or few) of which can be recognized. The co-PI's have collaborated in recent years on conformational analyses of a galactose-terminated triantennary oligosaccharide structure using fluorescence detected resonance energy transfer and found that individual conformations could be resolved using nanosecond time-resolved methods. We plan to launch a larger scale investigation to study conformational structure of carbohydrates of a wide variety of oligosaccharides terminated in galactose, N-acetyl- galactosamine, N-acetyl-glucosamine, and mannose. The conformational analysis of oligosaccharides will be examined in solution under a variety of conditions as well as when they are bound to purified receptor proteins. In addition new methodology will be developed to extend the studies to the oligosaccharides bound to the native receptors on the cell surface of hepatocytes. The investigation includes development of new chemical and enzymatic methods for modifying natural and synthetic carbohydrate ligands for addition of fluorescent probes. Special efforts will be directed at conjugation of the oligosaccharides with red- absorbing and emitting energy transfer pairs so as to decrease interference by auto-fluorescence of cells. Fluorescent oligosaccharide ligands will be used for studies of the mechanism of carbohydrate-protein interactions. In addition to the resonance energy transfer experiments, the studies will include emission anisotropy measurements to ascertain the amount of rotational flexibility of the carbohydrates on the protein and on the cell receptor sites. Fluorescence quenching will be used to determine the accessibility of the carbohydrate at the receptor sites. Other known photophysical events such as solvent relaxation and excited- state proton transfer will also be used to probe the character of the receptor sites.
