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Time-resolved tryptophan fluorescence anisotropy investigation of bacteriophage M13 coat protein in micelles and
K P Datema1, A J Visser, A van Hoek
1Department of Molecular Physics, Agricultural University, Wageningen, The Netherlands.
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 anisotropy 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 degrees C) and has a temperature dependence that satisfies the Stokes-Einstein relation. In vesicles 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 degrees 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 a 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.
Insights
Bacteriophage M13 coat protein structure changes in different environments. In vesicles, the protein forms beta-structures and aggregates, limiting tryptophan movement.
Area of Science:
- Biophysics
- Structural Biology
- Protein Dynamics
Background:
- The M13 bacteriophage coat protein is essential for viral assembly and infection.
- Understanding its structural dynamics in different membrane environments is crucial for deciphering its function.
Purpose of the Study:
- To investigate the secondary structure and dynamic behavior of the M13 coat protein.
- To elucidate the protein's interactions within lipid micelles and vesicles.
Main Methods:
- Time-resolved tryptophan fluorescence and anisotropy decay measurements.
- Circular dichroism spectroscopy.
- Analysis of protein-lipid interactions in various membrane models.
Main Results:
- Coat protein exhibits alpha-helical and beta-structures in sodium dodecyl sulfate micelles.
- Predominantly beta-structure is observed in lipid vesicles, suggesting conformational changes.
- Protein aggregation in vesicles restricts tryptophan indole ring motion, independent of lipid phase transitions.
Conclusions:
- The M13 coat protein undergoes significant structural changes depending on the membrane environment.
- Protein-protein aggregation in vesicles plays a key role in modulating protein dynamics.