The immunogenic properties of the P2 protein are known to be conformation dependent. A study of the tertiary structure of the molecule is therefore critical to an understanding of how the molecule participates in the disease induction process and of what structures and conformations are required of natural or synthetic peptides useful in protecting against induction of EAN or promoting recovery from the disease. Such information would have relavence for our understanding of human Guillian-Barre Syndrome and would provide us with the means to significantly improve treatment of that disorder. Exposed and buried regions of the P2 protein sequence will be identified in double-label experiments using reductive methylation to radio-methylate Epsilon-amino groups of lysines that are accessble under native (14C) and denatured (3H) conditions. In corrobarative studies, P2 protein will be subjected to chemical modification at exposed lysine residues (reductive Methylation and acetamidation) and the residual trypsin-sensitive lysines will identify buried sections of the molecule. Lactoperoxidase catalyzed iodination of the two P2 tyrosine residues will provide an independent assessment of exposure for regions of the modecule occupied by the amino and carboxyl terminal segments of the amino acid sequence. To determine which regions of the known primary structure are juxtaposed in the folded protein, P2 protein will be reacted with bis-alkylimidates and the regions joined in intramolecular lysine crosslinks indentified from analyzing peptides generated by cleaving at arginines with trypsin after blocking residual lysines with methylacetamidate. The above studies will make it possible to construct a model for the tertiary structure of the P2 protein. By studying the P2 protein complexed with phospholipids with the above techniques, it will be possible to determine changes in conformation induced by interaction with lipids. Using reductive radio-methylation on intact myelin, insights will be obtained into the relationship between conformations assumed by the P2 protein in myelin and in solution.
| Weise, M J (1986) A microcomputer program for hydropathic analysis of proteins with I/O through word processing and graphics software. Comput Appl Biosci 2:103-6 |
