Local dynamics of the M13 major coat protein in different membrane-mimicking systems.
The local environment of the transmembrane and C-terminal domain of M13 major coat protein was probed by site-directed ESR spin labeling when the protein was introduced into three membrane-mimicking systems, DOPC vesicles, sodium cholate micelles, and SDS micelles. For this purpose, we have inserted unique cysteine residues at specific positions in the transmembrane and C-terminal region, using site-directed mutagenesis. Seven viable mutants with reasonable yield were harvested: A25C, V31C, T36C, G38C, T46C, A49C, and S50C. The mutant coat proteins were indistinguishable from wild type M13 coat protein with respect to their conformational and aggregational properties. The ESR data suggest that the amino acid positions 25 and 46 of the coat protein in DOPC vesicles are located close to the membrane-water interface. In this way the lysines at positions 40, 43, and 44 and the phenylalanines at positions 42 and 45 act as hydrophilic and hydrophobic anchors, respectively. The ESR spectra of site specific maleimido spin-labeled mutant coat proteins reconstituted into DOPC vesicles and solubilized in sodium cholate or SDS indicate that the local dynamics of the major coat protein is significantly affected by its structural environment (micellar vs bilayer), location (aqueous vs hydrophobic), and lipid/protein ratio. The detergents SDS and sodium cholate sufficiently well solubilize the major coat protein and largely retain its secondary structure elements. However, the results indicate that they have a poorly defined protein-amphiphilic structure and lipid-water interface as compared to bilayers and thus are not a good substitute for lipid bilayers in biophysical studies.
Stopar
D
D
Department of Molecular Physics, Agricultural University, Wageningen, The Netherlands.
Spruijt
R B
RB
Wolfs
C J
CJ
Hemminga
M A
MA
D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins; D019826:Cholic Acid; D002793:Cholic Acids; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate
D036022:Capsid Proteins; D002793:Cholic Acids; D008565:Membrane Proteins; D008823:Micelles; D010713:Phosphatidylcholines; C025668:coat protein, Bacteriophage M13; D012967:Sodium Dodecyl Sulfate; C017251:1,2-oleoylphosphatidylcholine; D019826:Cholic Acid; D003545:Cysteine