| When creating a biosensor based on single ion channel activity, the conformational changes within a protein as well as the protein's ability to reconstitute successfully into non-native lipid environment and remain active must be understood. Therefore, the proximity of key residues in the C-terminal region of the Mechanosensitive Channel of Large Conductance (MscL) was quantified via disulfide bridging of cysteine mutations made to conserved hydrophobic residues in the linker and S3-bundle of this region. The biochemical assays utilizing disulfide bridging provided insight into which residues were dynamic and interactive, enabling them to be studied further via patch clamping. The channel's C-terminal region during gating was under debate and our results support the theory of the S3-bundle remaining closed during gating. Results from this study also enabled the generation of two new mutant channels that could coordinate heavy metals and the recognition response was a decrease in conductance, and slowed channel kinetics.;After the correct configuration of the protein was established, the ideal lipid environment for studying single ion channels in a tethered device for biosensor applications was investigated. The tip-dip electrophysiology method was used to determine an electrically stable lipid environment between different diphytanoyl compositions. Diphytanoyl lipids were vital components of this research due to their increased durability and stability when tethering to solid supports such as the gold surface on the device. Results indicated all of the lipid-ratios suffered from pore formation due to electrical breakdown, both reversible and irreversible. This was the first time that electroporation was reported at such low potentials as 125 mV and 40 mV which may be characteristic of using the tip-dip method with diphtanoyl lipids. The pore formation was random; however distinguishable from single ion channel conductance by configuring I-V curves and evaluating the kinetics of the pores formed in comparison to channel activity. The lipid ratio of 70PC/30PE was chosen as the most stable lipid ratio and was integrated as the synthetic lipid environment for both Gramicidin and MscL on the tBLM device. Results for Gramicidin, indicated that single channel activity within the tBLM were characteristic of the channel. On the other hand, a high applied voltage was required to gate MscL and the conductance response was lower for this channel in comparison to when it is in its natural environment. This lead to the incorporation of a voltage sensitive MscL mutant, K31E, which sensed tension when voltages as low as 85 mV were applied. Preliminary results indicate that this mutant is very active within the tBLM and single channel activity is attainable. |
