This application is for an administrative supplement to parent grant R01 GM108043 to request an instrument for conducting transport assays in an efficient and automated manner. The SURFE2R N1 from Nanion Technologies is the first commercial instrument to implement a Solid Supported Membrane (SSM) with capacitive coupling for measurement of transport from reconstituted proteoliposomes. In this case, proteoliposomes will contain KdpFABC, and transport assays with the SURFE2R N1 will complement other in vitro functional assays to characterize site-specific mutants in order to address mechanistic questions posed in the parent grant application: i.e., the path that K+ ions take as they traverse the molecule, elements of the protein complex that are responsible for energetic coupling, whether additional ions (e.g., H+) are co- transported, and if so with what stoichiometry. The SURFE2R N1 uses SSM technology to overcome the low flux rate innate to transporters and pumps by adsorbing large numbers of molecules to the relatively large (3mm) sensor and using capacitive coupling to measure their combined ion flow via high-gain/low-noise amplifiers routinely employed for patch-clamp technologies. Very low amounts of protein are required (0.1-1?g) and the automation of microfluidics provides not only for high time resolution, but also for programmed series of experiments to be run without user intervention. Indeed, once loaded with sample, a given sensor can be used for up to 100 individual experiments. Our current protocol using a stirred cuvette and a conventional fluorimeter requires 25?g of protein for each individual experiment with manual substrate additions and sample exchange. Thus, the SURFE2R N1 will provide a dramatic gain in throughput. In addition, data analysis software is included with the SURFE2R N1 which is far more sophisticated than the home-made analysis software currently applied to data from the fluorimeter. The availability of real-time data from the SURFE2R N1 with high time resolution allows fast binding kinetics to be resolved and routine determination of rate constants, EC50 and IC50. The performance of this instrument, which was not available at the time of the original application, has been validated on over 100 diverse targets, most of which are either pumps or secondary transporters. In preliminary trials on KdpFABC, our samples produced very robust signals that readily led to a precise ATP titration curve. Based on the suitability of our application, Nanion approved our application for a six-month loan of a SURFE2R N1 and a brand new instrument was recently shipped to our laboratory, where it is awaiting installation. This instrument would be available for immediate purchase should this grant application be approved. The SURFE2R N1 will also be useful for studies of the secondary transporter YiiP under another R01 grant from Dr. Stokes (GM125081) and a second cross-referenced application for this administrative supplement program has been submitted in order to share the expense.
The requested instrument will greatly facilitate measurements of potassium transport by the potassium pump KdpFABC and allow us to address mechanistic questions about how the potassium moves across the membrane and to identify structural elements necessary for this energy-dependent process.
| Huang, Ching-Shin; Pedersen, Bjørn Panyella; Stokes, David L (2017) Crystal structure of the potassium-importing KdpFABC membrane complex. Nature 546:681-685 |
