Myelin basic protein (MBP) is one of the major protein components of the CNS myelin sheath. The protein is closely associated with the inner folds of the myelin membrane where it provides stability to the multilamellar structure. Autoimmune responses to MBP have been implicated in the demyelinating diseases experimental allergic encephalomyelitis and multiple sclerosis. The exact mechanism of the interaction between MBP and the myelin membrane is unknown, but it has been suggested that the methylation of the protein on a specific arginine residue (Arg-108) is an important aspect of this interaction. This residue is methylated in a reaction with S-adenosylmethionine (AdoMet) by the enzyme AdoMet:MBP methyltransferase which we have recently isolated and kinetically characterized. In order to further characterize this important methylation reaction, a series of peptides will be synthesized which contain the sequence surrounding the methylated arginine residue in myelin basic protein. The kinetic mechanism for the methylation of these peptides by the enzyme will be deduced by steady state methods. Free energies for binding of the peptides to the enzyme will be determined. The product distribution and the rate of production of mono- and dimethylated products will be followed by analytical HPLC in order to determine if monomethylarginine peptides are released into solution. The stereospecificity of the methylation with respect to the chirality at sulfur will be investigated as will the possible role of sulfurane intermediates in transmethylations involving AdoMet. The solution dynamics and conformations of the synthetic peptides will be investigated by following T1 and T2 relaxations using spin-echo NMR methods and by 2-dimensional nuclear Overhauser enhancement spectroscopy (NOESY). The role of the methylation of the arginine residue on the conformation and binding properties of the peptides will be investigated. Specific interactions of the methylarginine residue with structural units (the """"""""serotonin site"""""""" and the """"""""hairpin loop"""""""") within the synthethic peptides and with MBP itself will be investigated by NMR spectroscopy. Methods for the large-scale purification of the enzyme will be developed and anti-methyl- transferase antibodies will be produced for the immunohistochemical localization of the enzyme within CNS cell- types. The work outlined in this proposal will directly contribute to our understanding of the chemical mechanisms and biological roles of transmethylation reactions involving AdoMet as methyl donor and will provide direct evidence concerning the existence and the importance of microscopic units of secondary structure within myelin basic protein.
