Configurations of the N-terminal amphipathic domain of the membrane-bound M13 major coat protein.
The M13 major coat protein has been extensively studied in detergent-based and phospholipid model systems to elucidate its structure. This resulted in an L-shaped model structure of the protein in membranes. An amphipathic alpha-helical N-terminal arm, which is parallel to the surface of the membrane, is connected via a flexible linker to an alpha-helical transmembrane domain. In the present study, a fluorescence polarity probe or ESR spin probe is attached to the SH group of a series of N-terminal single cysteine mutants, which were reconstituted into DOPC model membranes. With ESR spectroscopy, we measured the local mobility of N-terminal positions of the protein in the membrane. This is supplemented with relative depth measurements at these positions by fluorescence spectroscopy via the wavelength of maximum emission and fluorescence quenching. Results show the existence of at least two possible configurations of the M13 amphipathic N-terminal arm on the ESR time scale. The arm is bound either to the membrane surface or in the water phase. The removal or addition of a hydrophobic membrane-anchor by site-specific mutagenesis changes the ratio between the membrane-bound and the water phase fraction.
Meijer
A B
AB
Department of Biomolecular Sciences, Laboratory of Biophysics, Wageningen University and Research Center, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
Spruijt
R B
RB
Wolfs
C J
CJ
Hemminga
M A
MA
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000737:chemistry* / Q000235:genetics; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D036022:Capsid Proteins; D003497:Cyclic N-Oxides; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010713:Phosphatidylcholines / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013113:Spin Labels
D036022:Capsid Proteins; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D008567:Membranes, Artificial; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D013113:Spin Labels; C025668:coat protein, Bacteriophage M13; C024516:N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide; C006421:1,5-I-AEDANS; C017251:1,2-oleoylphosphatidylcholine; D003545:Cysteine