Structural characterisation and membrane insertion of M13 procoat, M13 coat and Pf3 coat proteins

Membrane proteins fulfill many central functions in the biological membrane. The insertion process of these proteins and their structure, which are intimately linked to their function, are not yet well understood. As a model we studied three proteins reconstituted into lipid bilayers (which serve as a model for the biological membrane): the major coat proteins of phages Pf3 and M13 and the precursor of the latter, M13 procoat protein, which spans the inner membrane two times and serves as a model for a multi-spanning membrane protein. Whereas Pf3 coat protein can insert into the membrane in its mature form, M13 coat protein needs the signal sequence present in the precursor to insert. A new, simple and efficient purification method has been developed for M13 procoat, M13 and Pf3 coat proteins. Homogenous preparations were obtained in isopropanol/0.l% TFA, where M13 coat protein is found to be dissolved in a monomeric form, and the two other proteins as dimers. The conformations of these particular proteins in different environments have been determined by circular dichroism and infrared spectroscopy. In organic solvents, the proteins adopt a conformation with an average helix content of 90%. In lipid bilayers composed of phospatidylcholine and phosphatidylglycerol lipids, the average helix content is 50% for M13 procoat protein, 60% for M13 coat protein and 75% for Pf3 coat protein. In order to characterize the orientational distribution the orientational order parameter Sα of the protein helices in planar lipid bilayers have been determined by polarized infrared measurements in the amide I spectral range. The helices of the three proteins are oriented preferentially parallel to the membrane normal, with Sα = 0.63 for M13 procoat protein, Sα = 0.58 for Pf3 coat protein and a distinctly higher value of Sα = 0.81 for M13 coat protein. The topology of M13 and Pf3 coat proteins was investigated using limited proteolysis in lipid vesicles with proteinase K combined with mass spectroscopy to analyze the lytic fragments. The C-terminal part of both proteins was found to be inaccessible to proteinase K, only 14 (Pf3) or 15 (M13) residues on the N-terminal were protease accessible from the outside of the vesicles. Furthermore, the standard free energy change, ΔGo, of a membrane-inserting protein with a leader sequence has been determined experimentally for the first time, using M13 procoat protein as an example. The partition coefficient for the distribution of this protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of Γ = 6.5×105 M-1, corresponding to a ΔGo of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC. For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: Γ = 1×105 M-1 (ΔGo = -9.