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ESR of spin-labeled bacteriophage M13 coat protein in mixed phospholipid bilayers
H H de Jongh1, M A Hemminga, D Marsh
1Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, F.R.G.
Abstract:
Bacteriophage M13 major coat protein was spin-labeled with a nitroxide derivative of iodoacetamide, preferentially at the single methionine that is located in the hydrophobic region of the protein. The spin-labeled protein was incorporated at different lipid-to-protein ratios in phospholipid bilayers composed of dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), or the 1:1 molar mixture of these lipids. Both conventional and saturation transfer (ST) ESR studies were performed to investigate the rotational motions of the protein over a large dynamic range. The conventional ESR spectra indicate that the mobility of the spin labelled protein in the lipid gel phase decreases in the order: DMPG greater than DMPC/DMPG (1:1) greater than DMPC. In the liquid crystalline phase, the largest mobility is found in DMPC/DMPG (1:1, mol/mol) mixtures, but the mobility is still greater in DMPG than in DMPC. The results are interpreted in terms of different degrees of protein aggregation in the different lipids. Segmental motion with rotational correlation times on the order of tens of nanoseconds, motional anisotropy, and spectral overlap complicate the analysis of the STESR spectra. An estimate of the size of the protein aggregates is found to be in the region of 85 monomer units. Removing the polar tails from the protein by proteolytic digestion results in an enhanced aggregation in the gel phase. In the liquid crystalline phase, the segmental wobbling mobility of the protein is increased relative to the native protein, whereas the overall rotational diffusion is not changed greatly.
Insights
Spin-labeling bacteriophage M13 major coat protein revealed its mobility in phospholipid bilayers. Protein aggregation varied with lipid composition, influencing rotational motion and aggregate size.
Area of Science:
- Biophysics
- Structural Biology
- Membrane Proteins
Background:
- Bacteriophage M13 major coat protein is a key component of viral structure.
- Understanding its behavior within lipid bilayers is crucial for membrane protein dynamics.
- Spin-labeling Electron Spin Resonance (ESR) is a powerful technique for studying protein motion.
Purpose of the Study:
- To investigate the rotational motions of spin-labeled M13 major coat protein in different phospholipid environments.
- To determine how lipid composition affects protein aggregation and mobility.
- To estimate the size of protein aggregates formed within lipid bilayers.
Main Methods:
- Spin-labeling of M13 major coat protein with a nitroxide derivative at the methionine residue.
- Incorporation of labeled protein into dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylcholine (DMPC) bilayers.
- Analysis using conventional and saturation transfer Electron Spin Resonance (ST-ESR) spectroscopy.
Main Results:
- Protein mobility in the gel phase decreased in the order: DMPG > DMPC/DMPG > DMPC.
- In the liquid crystalline phase, mobility was highest in DMPC/DMPG mixtures, followed by DMPG, then DMPC.
- Protein aggregation was influenced by lipid type, with aggregate sizes estimated around 85 monomer units.
- Proteolytic removal of polar tails enhanced aggregation in the gel phase and increased segmental wobbling in the liquid crystalline phase.
Conclusions:
- Lipid composition significantly impacts M13 major coat protein aggregation and rotational dynamics.
- Protein aggregation state is a critical determinant of its mobility within phospholipid bilayers.
- ST-ESR provides insights into protein aggregate size and segmental motion within membranes.