This application is for an administrative supplement to parent grant R01 GM125081 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 our case, these proteoliposomes will contain YiiP, which is the subject of the parent grant. Transport assays with the SURFE2R N1 will complement in vitro binding assays to characterize site-specific mutants of YiiP in order to address mechanistic questions posed in the parent grant application: i.e., the roles of the three different Zn2+ binding sites on the transport mechanism and on the energetic coupling that employs the proton-motive force to drive Zn2+ transport. Specifically, this machine will allow us to evaluate coupling stoichiometry (protons transported per Zn2+ ion) and the effects of site-specific mutations on this stoichiometry. The SURFE2R N1 uses SSM technology to overcome the low flux rate innate to secondary transporters by adsorbing large numbers of molecules to the relatively large (3mm) sensor and by 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 automation of microfluidic apparatus provides not only for high time resolution, but also for programmed series of experiments that run without user intervention. Once loaded with proteoliposomes, a given sensor can be used for up to 100 individual experiments. Thus, this machine provides a huge performance gain compared to our current protocol that relies on a conventional stop-flow fluorimeter. In particular, our current protocol requires 10x more protein and does not allow for changes in proteoliposome buffer conditions. In addition, 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 using data analysis software included with the SURFE2R N1, which is far more sophisticated than the home-made analysis software currently used to analyze data from the fluorimeter. The utility of the SURFE2R N1, 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. Our samples of YiiP were tested at the Nanion factory, producing robust signals that reflected both steady state transport and pre-steady-state binding events. As a result, an application for loan of this instrument was approved and Nanion recently shipped a brand new SURFE2R N1 to our laboratory, where it is awaiting installation. This unit would be available for immediate purchase should this request be approved. The SURFE2R N1 is also highly effective in quantifying ATP-driven K+ transport by KdpFABC, which is the subject of a separate R01 grant from Dr. Stokes (GM108043) and a second cross-referenced application has been submitted to share the purchase.
Our goal is to understand the mechanism by which the protein called YiiP transports zinc across biological membranes. The requested instrument will greatly facilitate our ability to measure zinc transport. These studies will allow us to address mechanistic questions about how this protein works and to identify structural elements necessary for this energy-dependent process.
