Time-resolved tryptophan fluorescence anisotropy investigation of bacteriophage M13 coat protein in micelles and mixed bilayers

Abstract

Coat protein of bacteriophage M13 is examined in micelles and vesicles by time-resolved tryptophan fluorescence and anisotropy decay measurements and circular dichroism experiments. Circular dichroism indicates that the coat protein has .alpha.-helix (60%) and .beta.-structure (28%) in 700 mM sodium dodecyl sulfate micelles and predominantly .beta.-structure (94%) in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidic acid (80/20 w/w) small unilamellar vesicles. The fluorescence decay at 344 nm of the single tryptophan in the coat protein after excitation at 295 or 300 nm is a triple exponential. In the micelles the anistropy decay is a double exponential. A short, temperature-independent correlation time of 0.5 .+-. 0.2 ns reflects a rapid depolarization process within the coat protein. The overall rotation of the coat protein-detergent complex is observed in the decay as a longer correlation time of 9.8 .+-. 0.5 ns (at 20.degree. C) and has a temperature dependence that satisfies the Stokes-Einstein relation. In veiscles at all lipid to protein molar ratios in the range from 20 to 410, the calculated order parameter is constant with a value of 0.7 .+-. 0.1 from 10 to 40.degree. C, although the lipids undergo the gel to liquid-crystalline phase transition. The longer correlation time decreases gradually on increasing temperature. This effect probably arises from an increasing segmental mobility within the coat protein. The results are consistent with model in which the coat protein has a .beta.-structure and the tryptophan indole rings do not experience the motion of the lipids in the bilayer because of protein-protein aggregation.