The interaction of M13 coat protein with lipid bilayers : a spectroscopic study

In this thesis a small part of the reproductive cycle of the M13 bacteriophage is studied in more detail, namely the interaction of the major coat protein (MW 5240) with lipid bilayers. During the infection process is the major coat protein of M13 bacteriophage stored in the cytoplasm membrane of the <em>E. coli</em> cell, while DNA replication takes place in the cytoplasm. The M13 coat protein in model lipid bilayers is present in two forms, which were previously called the alfa-oligomeric (b-state) and the β-polymeric (c-state) form. The secondary structure of the strongly aggregated M13 coat protein in the β-polymeric form is dominated by a high percentage of β-sheet conformation whereas the non-aggregated M13 coat protein in the α-oligomeric form was previously estimated to consist of 50% α-helix.A more precise knowledge of the secondary structure of the M13 coat protein in both forms helps to understand how protein-lipid interactions take place. Therefore, Circular Dichroism, Raman and Fourier Transform Infrared spectroscopy has been applied on lipid M13 coat protein systems reconstitutes. Care was taken to study either the aggregated or non aggregated form of the M13 coat protein. The β-polymeric form of M13 coat protein has a secondary structure of 13% α-helix, 57% β-sheet, 13% turn and 16% remainder and the α-oligomeric M13 coat protein is in a 91% α-helix, 5% β-sheet, 3% turn and 1% remainder conformation. The overall conformation of the M13 coat protein in the α-oligomeric form corresponds well with the conformation of virion bound coat protein.In a more theoretical study it was tried to relate the conformation and aggregation state of the M13 coat protein. MD simulations were performed on M13 coat protein in the β-polymeric and the α-oligomeric form. The simulations were started from initial conformations of M13 coat protein as monomers or dimers of alfa-helices or U-shaped β-sheets. The M13 coat protein in the U-shaped βstructure changes from a planar to a twisted form with larger twist for the M13 coat protein as monomer than as a dimer. The M13 coat protein in the beta-sheet conformation β-polymeric is much more flexible than the M13 coat protein in an a-helix conformation (α-oligomeric) as monitored by the rms fluctuations of the Cαatoms. A comparison of the energies after 100 ps MD simulation shows that of the monomers, M13 coat protein in an a-helix has the lowest energy. The energy difference between α- and β-structures decreases from 266 kJ/mol to 148 kJ/mol when going from monomers to dimers. It is expected that this difference will decrease with higher aggregation numbers, suggesting that the M13 coat protein in the β-sheet, conformation must be aggregated, in agreement with observations performed with HPLC.Using <sup>2</SUP>H-NMR and <sup>31</SUP>P-NMR the interaction of the M13 coat protein in both forms with specific headgroup and chain deuterium labelled phospholipids is studied. It can be concluded from the spectra that the protein in the predominant βsheet conformation causes a fraction of lipids to be trapped. Together with the information obtained from the structure and aggregation state of this form (chapters 2 and 3) it is suggested that the trapped lipids are arranged in a non bilayer structure, probably induced by a misfitting of the hydrophobic core of the protein and the membrane bilayer. The protein in the predominant a-helix conformation perfectly fits in the lipid bilayer and has only minor influences on the surrounding lipid matrix.However, because in the inner membrane only α-helical proteins are found, the correspondence with the conformation of the virion bound protein and the fact that the aggregation of the β-polymeric protein is irreversible it is suggested that the α-oligomeric protein is the more likely form of the M13 coat protein to be found in the <em>E. coli.</em> Therefore, a more detailed study was performed on the interaction of this form of the M13 coat protein with lipid bilayers. The <sup>2</SUP>H-NMR quadrupolar splittings of the αhead-group methylene deuterons of deuterated phosphatidylcholine and phosphalidylethanolamine decrease, whereas the quadrupolar splittings of the βhead-group methylene deuterons of the two lipids increase with increasing protein content. All deuterated segments in the phosphatidylglycerol headgroup show the same relative decrease of the NMR quadrupolar splittings. These results are interpreted in terms of a change in torsion angles of the methylene groups, induced by positive charges, probably lysine residues of the protein at the membrane surface. For all lipid bilayer compositions studied the head-group perturbations are similar. It is concluded that there is no strong specific interaction between one of the lipid types examined and the M13 coat protein.In the ESR spectra of spin labels in lipid bilayers no second protein induced component can be observed upon incorporation of M13 coat protein in the α-oligomeric form. It is argued on the bases of ESR and time resolved fluorescence results that M13 coat protein in the α-oligomeric does not immobilize lipids but restricts the order and motion of the lipids, due to its cylindrical form, proportions and non specific interaction with lipids in the bilayer.Finally, from this work it can be concluded that the influences of the M13 coat protein in its probably natural form, the α-oligomeric form, on the lipid membranes are small. Positive charges are introduced at the bilayer surface and the hydrophobic area is less fluid in the presence of the M13 coat protein. This let us to understand that higher amounts of cardiolipin are produced when M13 is infecting <em>E. coli.</em> M13 coat protein changes the charge density at the membrane surface which is counteraffected by cardiolipin. In addition the machinery of <em>E. coli</em> is also capable in retaining the fluidity of its membranes simply by changing the lipid tail composition. This let us understand that even high concentrations of M13 coat protein are not lethal for its host, <em>E. coli.</em>