Isolation and Use of the Aeromonas hydrophila Secretin ExeD for Nanopore Analysis
Nanopore analysis is a technique for measuring single molecule interactions with an open pore in solution. Current progress in the field is hindered by the size of the most commonly used nanopore, α-hemolysin. The narrow vestibule of the pore doesn’t allow proteins or peptide aggregates to pass through its channel. A larger pore, using the type II secretin of Aeromonas hydrophila, may be able to circumvent the size constraint of α-hemolysin and could allow research into larger molecules to be made. The goal of this project was to isolate and use the type II secretin ExeD in nanopore analysis. After this was accomplished, the ExeD pore would be inserted into a lipid bilayer in a patch clamp setup where observations could be made about the ability of ExeD to translocate small molecules and peptides. If ExeD could translocate larger peptides than α-hemolysin, nanopore analysis could be expanded to include these larger molecules. Nanopore research has previously been done using the ExeD homologues PulD and XpcQ, but results were unclear due to contaminant proteins and problems with measuring the molecular interactions. In the case of PulD, the tightly-bound lipoprotein PulS co-purified with PulD and could not be separated prior to the experiments. To avoid the presence of a similar lipoprotein co-purifying with ExeD, the gene was cloned from the bacteria Aeromonas hydrophila (which does not have a PulS homologue) and expressed in E. coli. The protein was then purified using a combination of detergents and chromatography. The resultant extract was shown to be free from contaminant proteins and capable of inserting into a planar lipid bilayer. Previous research using XpcQ were inconclusive due to both an ion-leak across the membrane and from having uncertain pore insertions. Our experiments showed that under specific conditions, pore insertions could be consistently repeated and easily identified, and that ion-leak would not occur. We further identified a new method in inserting ExeD into lipid bilayers. Currently, membrane proteins often require proteoliposomes for inserting the pore into the lipid bilayer. Our research found that using an acetone precipitate of ExeD was preferable to forming liposomes due to both the ease of the protocol and the pore’s ability to insert from a precipitate. During the course of the study it was found that when the exeD gene was highly expressed using elevated temperatures and high amounts of the transcriptional inducer IPTG, the self-assembling protein would produce unusual results in patch clamp. The gating and conductance of these pores were highly variable and difficult to reproduce across experiments. We concluded that these pores were non-native conformations of the multimer and had been incorrectly assembled. It was found that at low temperatures and low levels of IPTG, protein expression not only increased but the pores which were produced were uniform and had identical activity to one another. The conformation of the protein that most likely represents the native form was identified from both its prevalence across a range of expression conditions, from its behavioural similarity to PulD, and from its channel characteristics. We compared gating events and pore conductance at three different pH levels and several voltages. At pHs of 5 and 7.4 gating is rapid and the events fall into a characteristic pattern depending on the applied voltage. At a pH of 9.5 gating is far less frequent and the events produce a smaller blockade current with a unique distribution from those of pH 7.4 and 5. It was found that using a pH of 9.5 and a low applied voltage lowered the natural gating levels considerably, making DNA interactions distinguishable from naturally occurring gating events and producing a peak which we believe corresponds to translocation. A small α-helical peptide was also examined under similar conditions, but produced only bumping interactions, showing that peptides are unable to translocate. These results demonstrate that ExeD is likely capable of passing strands of DNA through its channel, but molecules the size of peptides or larger may require channel activation from additional proteins. In conclusion, we have extensively studied the secretin ExeD and have found it is not suitable for studying peptides or proteins as it currently exists.
Nanopore Analysis, ExeD, Aeromonas hydrophila
Master of Science (M.Sc.)