My laboratory is interested in fundamental molecular mechanisms by which cells maintain ionic homeostasis. We study two particular systems responsible for transport of K+ and Zn2+, respectively. Overall goals are the same for both systems, which are to develop a comprehensive understanding of transport from structural as well as thermodynamic perspectives. We will use a broad spectrum of biophysical and biochemical approaches, including cryo-EM for structure determination, in vitro biophysical assays for functional characterization, single-molecule FRET and Molecular Dynamic simulations for analyzing dynamics of the molecules. In this way, we aim to define an energy landscape for each system, annotated with the experimental structures for stable intermediates as well as an appreciation for the high-energy transition states that define the transport pathway. We also seek to understand determinants of substrate specificity and structural elements responsible for the allosteric coupling that underlies energy coupling and regulatory mechanisms. The first system under investigation is KdpFABC, an interesting and unusual hybrid between an ATP-dependent pump related to P-type ATPases and a K+ channel related to the Superfamily of K+ Transporters. Our previous work has defined the architecture of this heterotetramer and suggests a highly novel mechanism for transport, in which K+ enters the selectivity filter of the channel-like subunit, travels 40 Å through a membrane-embedded tunnel, and is then expelled by the pump-like subunit in an energy-dependent manner. We now plan functional analyses of site-directed mutants to validate this hypothesis and to adopt new approaches to study the energetics. The second system is YiiP, a Zn2+/H+ antiporter from the Cation Diffusion Facilitator superfamily. Members of this family form homodimers, have multiple ion binding sites and are thought to function via an alternating access mechanism. For this system, we have characterized two different conformations by cryo-EM analysis and MD simulation, and have identified a role for Zn2+ binding on the transition. We seek to understand better the role of individual Zn2+ binding sites, the nature of occluded states, the transition between inward- and outward-facing states and the role of protons in this process. We plan also to study additional members of this family to address the basis for substrate specificity and structural features that have been postulated to regulate the transport process. In addition to shedding light on these two specific transport mechanisms, we hope that our work will offer new ways to think about transport that goes beyond cartoons and structural animations to incorporate protein dynamics and mapping of the energy landscape to describe the behavior of these molecular machines.