The principal goal of this research project is the determination of the tertiary structure of the diphtheria toxin using x-ray crystallographic analysis of their single crystals. The toxin is the protein responsible for the observed lesions associated with that disease. The protein is synthesized in the bacteria as a single polypeptide of molecular weight 60,000. The protein is made of two fragments. Fragment A has a molecular weight of 21,500 and it specifically inhibits protein synthesis by catalyzing the transfer of the ADP-ribose moiety of NAD+ to eukaryotic EF-2 factor in the cytoplasm. Fragment B has a molecular weight of 40,000 and is required for the recognition of specific surface receptors on the cell membranes. On cleavage of a single peptide bond and a disulfide bridge the toxin splits into the two fragments and the catalytic subunit is internalized. Crystals of the diphtheria toxin suitable for structure analysis have been grown by the Principal Investigator. The crystals belong to the trigonal space group P3(1)12 with unit cell dimensions of a equals b equals 97.9A and c equals 100.3A. There is one molecule of the toxin per asymmetric unit. The crystals diffract to a resolution of 2.8A and the structure analysis will be carried out using the multiple isomorphous replacement method. Interest in the structure analysis of the diphtheria toxin has been stimulated by numerous attempts to use the toxins as """"""""magic bullets"""""""" to kill malignant cells while leaving normal cells unharmed. This is achieved by preparing hybrid proteins containing the toxic component of the diphtheria toxin conjugated to proteins capable of binding to specific receptor sites on target cells. This has wide chemotherapeutic applications particularly in the treatment of cancer.
