The membrane-bound form of gene 9 minor coat protein of bacteriophage M13

Bacteriophage M13 is a virus that infects the bacteria Escherichia coli ( E. coli ), a single cell organism that resides in our intestines. It consists of the cytoplasm (contents) and a double membrane that keeps the contents together (the barrier to the outside world). The membrane is formed by lipid molecules which consist of a head group that very much likes water (hydrophilic) and two fatty tails that dislike water (hydrophobic). In order to avoid contact with water the fatty tails group together in such a way that a planar double layer (bilayer) of the lipid molecules results. In this lipid bilayer also proteins are present to fulfill different functions, e.g. regulate transport across this barrier. The lipids and proteins together form the membrane. However, this membrane is not a complete barrier for certain intruders in the E. coli cell. A virus, such as M13 bacteriophage, can enter the E. coli cell. It uses the E. coli cell as a host by using the cell's components to multiply itself. And finally the bacteriophage is able to cross the membrane and put itself together on its way out. During this latter process, called the assembly, the new bacteriophage is formed out of its components. It is a special feature of bacteriophage M13 that the assembly takes place in the membrane. Even more special is that bacteriophage M13 does not kill its host. Normally, many other viruses kill their hosts by lysing the cells. Bacteriophage M13 consists of a single DNA molecule, which codes the genomic information, and a "coat" of proteins, which surrounds and protects the DNA. Bacteriophage M13 looks like a rod of almost 1 micrometer length and about 6.5 nanometer diameter. The coat proteins along the long side are all identical and present in many copies (about 2700) and therefore are called major coat proteins. On either end of the bacteriophage are a few different coat proteins, called minor coat proteins. One of these is the topic of this work: the gene 9 protein (g9p). G9p consists of only 32 amino acids. It is located on the end of the bacteriophage that emerges first from the E. coli cell during the assembly. Therefore, g9p is expected to play a role early in the assembly process. Genetic experiments also showed that g9p is a possible candidate to interact with the tip of the DNA molecule. This makes g9p an interesting object of study, since it might lift up some of the veil over the assembly process. The assembly process is still like a black box: the components that go in and the outcome (intact bacteriophage) are known, but what happens in between is still a mystery, although parts of it have been revealed. By taking a closer look of g9p we hope to contribute a small piece in unraveling this mystery. Up to now the structure and position of g9p before assembly were based on speculation. In this thesis we aimed to characterize g9p in more detail by analysis of the structure it adopts in a membrane. In addition, we aimed to determine how and which side enters the membrane.ResearchTo study g9p we used chemically synthesized protein and since biological membranes are very complex structures we studied g9p in simpler artificial membranes.G9p has been predicted to be hydrophobic (Chapter 2) and is therefore difficult to handle. It is difficult to remove the protein once it sticks to a surface, and it has a tendency to form aggregates by sticking to each other. It can be dissolved in the organic solvent TFE. In an aqueous solution, detergents are necessary to solubilize the protein. In Chapter 2 conditions for solubilizing the protein were found. In a weak detergent g9p appeared to aggregate. This was accompanied by a conformational change froma-helix tob-sheet, as measured by CD and FTIR. The protein was incorporated in phospholipid membranes via TFE or detergent. When using detergent, care had to be taken to avoid aggregation intob-sheets. Phospholipid membranes appeared to preserve the state of the protein, either aggregated or non-aggregated. The work described in Chapter 2 resulted in the production of reproducible protein-lipid systems, which were necessary to study g9p in the membrane-bound state.Information about the structure of g9p is described in Chapter 2 and 3. CD and FTIR revealed that g9p is predominantlya-helical in the phospholipid membrane. By analysis of the amide I band of the FTIR spectrum secondary structure elements were quantified. About 67% of the protein structure wasa-helix, and the remaining part was assigned to be turn structure. In addition, the orientation of thea-helix axis with respect to the membrane normal was determined by measuring ATR-FTIR spectra of the protein incorporated in oriented bilayers. G9p appeared to be oriented preferentially parallel to the membrane normal, which means that the orientation is mainly transmembrane (Chapter 3). Based on these results and general principles that govern the positioning of protein domains in bilayers, we proposed possibilities for a tentative structural model of g9p in a membrane (Chapter 3 and 5). One model assumes a single N-terminal transmembrane helix of 21 amino acid residues long and a C-terminally located turn structure. An alternative model assumes a 15 amino acid trans-membrane helix and a second shorter amphipathic helix, which is more in line with the membrane plane. Turn structure links the two helices. The remaining turn structure is located C-terminally according to this model. This part was shown to contain the antigenic site (Chapter 4). The latter helix-turn-helix model remarkably resembles models of the major coat proteins of bacteriophage M13 and related bacteriophages.In Chapter 4 g9p was found to be able to insert spontaneously into phospholipid monolayers and bilayers. In addition, the N-terminal side of the protein was determined to be the part to insert, whereas the C-terminal part was determined to remain outside the membrane (N in -C out orientation). This was shown by cleavage experiments. After cleavage, the presence or absence of the antigenic site, which was found to be located at the C-terminal end of the protein, functioned as a determinant of the topology. Negatively charged phospholipids may enhance the efficiency of the adoption of a N in -C out orientation, which is in agreement with our experimental results and with literature (Chapter 5).The relevance of these experimental results with model systems is that they may provide indications for the in vivo situation. Up to now, no evidence was available regarding the conformation of g9p. The predominantlya-helical conformation we observed seems likely to be present in vivo also (Chapter 5). The finding that g9p can insert spontaneously in model membranes may be relevant for the in vivo situation. Our orientational and topological results show what part of the protein is buried in the membrane and what part is accessible. Translated to the in vivo situation this implies that the C-terminal side is sticking out in the cytoplasm, thus making it a good candidate to interact with the viral DNA in the assembly process.