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Membrane location of spin-labeled M13 major coat protein mutants determined by paramagnetic relaxation agents
1Department of Molecular Physics, Agricultural University, Dreijenlaan 3, NL-6703 HA Wageningen, The Netherlands.
Abstract:
Mutants of the M13 bacteriophage major coat protein containing single cysteine replacements (A25C, V31C, T36C, G38C, T46C, and A49C) in the hydrophobic and C-terminal domains were purified from viable phage. These were used for site-directed spin-labeling to determine the location and assembly of the major coat protein incorporated in bilayer membranes of dioleoylphosphatidylcholine. The membrane location of the spin-labeled cysteine residues was studied with molecular oxygen and Ni2+ ions as paramagnetic relaxation agents preferentially confined to the hydrophobic and aqueous regions, respectively, by using progressive-saturation electron spin resonance (ESR) spectroscopy. The section of the protein around Thr36 is situated at the center of the membrane. Residue Thr46 is placed at the membrane surface in the phospholipid head group region with a short C-terminal section, including Ala49, extending into the aqueous phase. Residue Ala25 is then positioned consistently in the head group region of the apposing lipid monolayer leaflet. These positional assignments are consistent with the observed mobilities of the spin-labeled groups. The outer hyperfine splittings in the ESR spectra decrease from the N-terminal to the C-terminal of the hydrophobic section (residues 25-46), and then drop abruptly in the aqueous phase (residue 49). Additionally, the strong immobilization and low oxygen accessibility of residue 25 are attributed to steric restriction at the hinge region between the transmembrane and N-terminal amphipathic helices. Sequence-specific modulations of the ESR parameters are also observed. Relatively low oxygen accessibilities in the hydrophobic region suggest intermolecular associations of the transmembrane helices, in agreement with saturation transfer ESR studies of the overall protein mobility. Relaxation enhancements additionally reveal a Ni2+ binding site in the N-terminal domain that is consistent with a surface orientation of the amphipathic helix.
Insights
Site-directed spin-labeling determined the M13 bacteriophage major coat protein
Area of Science:
- Biophysics
- Structural Biology
- Molecular Biology
Background:
- The M13 bacteriophage major coat protein plays a crucial role in phage assembly and membrane integration.
- Understanding the precise location and orientation of this protein within lipid bilayers is essential for elucidating its function.
- Previous studies have provided limited information on the detailed structural organization of the coat protein in a membrane environment.
Purpose of the Study:
- To determine the precise location and assembly of the M13 bacteriophage major coat protein within dioleoylphosphatidylcholine bilayer membranes.
- To investigate the protein's structural organization using site-directed spin-labeling and electron spin resonance (ESR) spectroscopy.
- To map specific residues within the hydrophobic and C-terminal domains relative to the membrane interface.
Main Methods:
- Site-directed mutagenesis to introduce single cysteine residues at specific positions (A25C, V31C, T36C, G38C, T46C, A49C) in the M13 major coat protein.
- Purification of mutant phage and incorporation of labeled coat protein into dioleoylphosphatidylcholine bilayer membranes.
- Progressive-saturation electron spin resonance (ESR) spectroscopy utilizing molecular oxygen and Ni2+ ions as paramagnetic relaxation agents to probe membrane location.
Main Results:
- Residue Thr36 was localized to the center of the lipid bilayer.
- Residue Thr46 was found at the membrane surface within the phospholipid head group region, with a short C-terminal extension (Ala49) into the aqueous phase.
- Residue Ala25 was positioned in the head group region of the opposing lipid monolayer, indicating specific interactions and potential steric restrictions.
Conclusions:
- The study successfully mapped key residues of the M13 major coat protein within a model membrane system.
- The findings suggest a specific orientation and assembly of the coat protein, with transmembrane and amphipathic helical regions.
- Evidence for intermolecular associations within the hydrophobic core and a surface-oriented N-terminal amphipathic helix was observed.