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Conventional and saturation-transfer EPR of spin-labeled mutant bacteriophage M13 coat protein in phospholipid
W F Wolkers1, R B Spruijt, A Kaan
1Department of Molecular Physics, Agricultural University, Wageningen, Netherlands.
Abstract:
A mutant of bacteriophage M13 was prepared in which a cysteine residue was introduced at position 25 of the major coat protein. The mutant coat protein was spin-labeled with a nitroxide derivative of maleimide and incorporated at different lipid-to-protein (L/P) ratios in DOPC or DOPG. The rotational dynamics of the reconstituted mutant coat protein was studied using EPR and saturation transfer (ST) EPR techniques. The spectra are indicative for an anisotropic motion of the maleimide spin label with a high order parameter (S = 0.94). This is interpreted as a wobbling motion of the spin label with a correlation time of about 10(-6) to 10(-5) s within a cone, and a rotation of the spin label about its long molecular axis with a correlation time of about l0(-7) s. The wobbling motion is found to correspond generally to the overall rotational motion of a coat protein monomer about the normal to the bilayer. This motion is found to be sensitive to the temperature and L/P ratio. The high value of the order parameter implies that the spin label experiences a strong squeezing effect by its local environment, that reduces the amplitude of the wobbling motion. This squeezing effect is suggested to arise from a turn structure in the coat protein from Gly23 to Glu20.
Insights
Researchers studied M13 bacteriophage coat protein dynamics using electron paramagnetic resonance (EPR). They found that the protein
Area of Science:
- Biophysics
- Structural Biology
- Membrane Proteins
Background:
- Bacteriophage M13 major coat protein is a model for studying protein-lipid interactions.
- Understanding coat protein dynamics is crucial for viral assembly and function.
Purpose of the Study:
- To investigate the rotational dynamics of M13 major coat protein reconstituted in lipid bilayers.
- To characterize the motion of a spin-labeled cysteine mutant at position 25.
Main Methods:
- Site-directed mutagenesis to introduce cysteine at position 25.
- Spin-labeling with a maleimide derivative.
- Reconstitution into dimyristoylphosphatidylcholine (DOPC) or dimyristoylphosphatidylglycerol (DOPG) lipid bilayers at varying lipid-to-protein ratios.
- Electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy.
Main Results:
- The spin label exhibited anisotropic motion with a high order parameter (S = 0.94).
- Two distinct motional components were identified: wobbling within a cone (10^-6 to 10^-5 s) and rotation about the molecular axis (10^-7 s).
- Protein motion was sensitive to temperature and lipid-to-protein ratio, indicating environmental influence.
Conclusions:
- The spin label's motion reflects the overall rotational dynamics of the coat protein monomer.
- A strong squeezing effect from the local environment restricts the wobbling motion.
- This squeezing may be attributed to a turn structure within the coat protein (Gly23-Glu20).