PROJECT SUMMARY/ABSTRACT Membrane transport proteins constitute an important part of our genome and are target to many therapeutic drugs, but the complexities inherent to their hydrophobicity have significantly delayed their biochemical and structural studies. ATP binding cassette (ABC) transporters use energy from ATP hydrolysis to move a large variety of molecules against their concentration gradient across the membrane. Humans have near 50 ABC transporters, but the function of many of them remains unknown. Such is the case of the ABCB10 transporter, located in the mitochondrial inner membrane, where it is overexpressed in failing human heart. Diverse experimental evidence indicate that this transporter is necessary for the synthesis of heme and for protection of erythropoietic and cardiac cells against oxidative stress, but the identity of its physiological substrate and its molecular mechanism of transport are uncertain. It has been proposed that ABCB10 exports heme out the mitochondrial matrix (where heme?s final synthesis steps occur), although no substantial evidence is available. Our preliminary studies indicate that a heme analog modulates the activity ABCB10, which constitutes the first in vitro evidence supporting the putative role of this transporter in heme transport. The goal of the first aim of this project is to understand how ABCB10 binds heme. Can ABCB10 bind other heme analogs (metalloporphyrin bound to different metals, which can be available in the cells under certain conditions)? or precursors (metal-free porphyrin)? What residues of the protein are important for this binding? Answering these questions is important to determine the substrate specificity of ABCB10 and can expose some of these molecules as modulators of the transporter (activators or inhibitors), whose relevance can be tested in future physiological studies. The goal of our second aim is to understand, at the molecular level, how ABCB10 is capable of coupling hydrolysis of ATP to substrate transport. Multiple experimental evidence (including structures obtained by X-ray crystallography and more recently, Cryo-electron microscopy) suggest that ABC transporters follow an alternating access mechanism, where the location and affinity of the substrate binding site change during ATP binding and hydrolysis. Here we will study the molecular events that lead to conformational changes in the transporter because this basic information is important to understand the function of the transporter and to predict how it can be deliberately modified with therapeutic purposes. Our approach consists on a combination of biochemical and spectroscopic methodologies, that star with the heterologous expression, detergent solubilization and purification of human ABCB10. In our experience, the structure and function of ABC transporters are significantly altered when these proteins are out of their membrane environment, so we reconstitute the purified transporter in small and soluble lipid bilayers known as nanodiscs. This constitutes an excellent preparation to study the effect of substrates on the ATPase activity of the transporter proposed in our first aim. Site directed mutagenesis will be used to determine the residues important for heme binding to the transporter. For the study of the conformational changes in aim two we will use Luminescence Resonance Energy Transfer (LRET), a spectroscopic technique that works as a molecular ruler to measure distances between probes attached to specific residues (cysteines) strategically positioned in a region of interest. The simultaneous use of nanodisc-reconstituted protein and LRET to monitor conformational changes in functional ABC transporters is an innovative approach developed by our group and which has proven to be extremely useful for our recent studies of the multidrug resistance abcb1 (P- glycoprotein) and its bacterial homolog MsbA. We have created a functional cysteine-less ABCB10, and we will introduce single cysteines along this homodimeric transporter to study conformational changes during its activity cycle. This proposal is significant because it will provide basic knowledge that is important to help understand the molecular mechanism of a human protein yet poorly understood, and which can be a potential target for treatment of anemia and cardiac protection against oxidative stress.