Fluorescence and protein structure: XIV. Tyrosine fluorescence in helical muscle proteins

Abstract

1. 1.|The muscle proteins paramyosin and tropomyosin were chosen for this study of the effect of helical conformation on fluorescence of Tyr residues because of their high helical content and the absence of tryptophan. 2. 2.|Aggregation of the proteins did not affect fluorescence. This suggested Tyr residues were not in the interface between aggregating monomers and probably the aggregation was essentially end-to-end. 3. 3.|Because of the current uncertainty of absolute quantum yields for tyrosine and Tyr residues, fluorescence has been expressed as the ratio, R Tyr, of observed fluorescence relative to that of l-tyrosine at pH 7. In these terms, the fluorescence at pH 7 for paramyosin ( R Tyr = 0.40) and tropomyosin ( R Tyr = 0.26) rose to a common value ( R Tyr = 0.67) at pH 2. Esterification of the carboxylate groups on the proteins demonstrated that the lower fluorescence at pH 7 was from partial quenching by nearby Asp or Glu residues. 4. 4.|Iodination of the protein Tyr residues indicated there were no energy transfers between residues as though the Tyr residues were widely spaced over the surface of these helical rods. 5. 5.|Loss of helical conformation, by thermal denaturation or by trypsin digestion, decreased the fluorescence to levels typical of Tyr residues in randomly coiled polypeptides ( R Tyr = 0.25–0.35). This lowered fluorescence was attributed to quenching by peptide carbonyl groups in the random coil but not in the α-helical conformation. 6. 6.|A classification of Tyr residues that is based on fluorescence has been revised to include these highly fluorescent residues in regions of α-helix.