Insane in the membrane: Insertion of marginally hyrdophobic transmembrane helices and global analysis of membrane protein topology

Sammanfattning: Proteins are responsible for carrying out most of the tasks in a living cell; transcription, translation, replication, movement, catalysis and communication to mention a few. A subset of proteins is the integral membrane proteins, IMPs, which play an important role in governing communication across membranes, whether it is the plasma membrane or an organelle membrane. As IMPs are often responsible for uptake of substances such as hormones and pharmaceuticals they are also very often prime targets in medical research. In order for an IMP to function correctly it must fold and insert into the membrane properly as well as display the correct motifs to its surroundings, on either side of the membrane. IMPs cannot spontaneously insert into the membrane, the insertion is assisted by the Sec-translocon machinery. The Sec-translocon creates a pore in the membrane enabling nascent polypeptides to translocate across the membrane. This thesis will cover both the insertion of IMP segments as well as an evaluation of different approaches on how to investigate the topology of integral membrane proteins.The first question to be addressed is whether there are any specific sequence motifs within the sequence context that can improve the co-translational insertion of a marginally hydrophobic transmembrane helix, mTMH, into the membrane. A mTMH is a protein segment that would not insert by itself into the membrane. It has however been shown that these mTMHs can insert effectively into the membrane using their neighboring helices and loops, referred to as its sequence context, to compensate for the unfavorable insertion of only the mTMH. We show for a number of mTMHs that disrupting the sequence context motifs, usually lowering the ?G-values for insertion by introducing more hydrophobic residues through substitutions in the sequence context, does not by itself improve the insertion of a given mTMH. It can however be concluded that the positive inside rule is of great importance to improve recognition and co-translational insertion of these mTMHs as it provides an oriental preference of the subsequent helix. This oriental preference will enable the mTMH to insert. This means that the positive inside rule it stronger when followed by a transmembrane helix, at least for the insertion of mTMHs.The second question addressed is that of how to design a method to analyze the topology of membrane proteins in a high-throughput proteomic fashion. In order to extract information on membrane protein topology a protease can be used to degrade the exposed parts of the integral membrane protein, known as shaving. These peptides can then subsequently be degraded and analyzed using MS and bioinformatics. To compare different proteases, we first apply our shaving experiment on two over-expressed proteins and analyze the detected peptides using MS. Secondly; we run the same experiment on non-over-expressed Escherichia coli membrane proteins with known structure. Finally, the results from the above experiments were used to test the accuracy of a number of topology predictors. We can conclude that the use of the protease Thermolysin does show promising results when compared to for instance trypsin. Even though the two proteases show somewhat similar output on the proteins used in this study, Thermolysin does produce fewer peptides originating from the transmembrane region. This is most likely due to the milder, more native like reaction conditions combined with the shorter incubation time used for Thermolysin as compared to trypsin. These properties are believed to greatly improve the output and accuracy when applied on large scale global analysis.