Conventional and saturation-transfer EPR of spin-labeled mutant bacteriophage M13 coat protein in phospholipid bilayers.
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.
Wolkers
W F
WF
Department of Molecular Physics, Agricultural University, Wageningen, Netherlands.
Spruijt
R B
RB
Kaan
A
A
Konings
R N
RN
Hemminga
M A
MA
D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D036022:Capsid Proteins; D004578:Electron Spin Resonance Spectroscopy; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry*; D011487:Protein Conformation; D013113:Spin Labels
D036022:Capsid Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010743:Phospholipids; D013113:Spin Labels; C025668:coat protein, Bacteriophage M13