Difluoromethionine as a novel F-19 NMR structural probe for internal aminoacid packing in proteins

The successful incorporation of difluoromethionine (DFM), a novel F-19 NMR probe of internal amino acid packing, into the three methionine positions ( 1, 14, and 107) of a recombinant protein, the lysozyme from bacteriophage l ambda (LaL), is reported. The anisochronous F-19 NMR signals of the diaster eotopic fluorines showed a variation in the degree of chemical shift differ ence when present at relatively free surface positions (Met1 and Met107) ve rsus the tightly packed protein core (Met14), with the anisochronicity grea tly enhanced for DFM incorporated at this latter position. The increased ma gnetic nonequivalence of the two fluorines at position 14 is thought to be a consequence of the restricted environment of DFM at this position. The an isochronicity of these two fluorines is further manifested in a differentia l chemical shift change for these two fluorines upon binding of an oligosac charide inhibitor to LaL, with one of the two fluorines experiencing a sign ificant upfield shift compared to the other. This differential variation is thought to be associated with a very subtle change in the protein conforma tion surrounding one fluorine at position 14, which is not significantly tr anslated to the environment of the other fluorine.