Apolipoprotein-B (apoB)-containing lipoproteins are both a biomarker and a causal mediator of many central hallmarks of metabolic disease, including insulin resistance, fatty liver disease, atherosclerosis, obesity and metabolic syndrome. Inhibition of microsomal triglyceride transfer protein (MTP), a heterodimeric complex of MTP and protein disulfide isomerase (PDI) subunits, profoundly reduces specifically atherogenic apoB- containing lipoproteins by 50%, but it causes hepatosteatosis and steatorrhea of the intestine. MTP complex transfers different lipids and assists in the production of apoB-containing lipoproteins. Our recent work provides the first evidence that the triglyceride (TG) and phospholipid (PL) transfer functions of MTP can be decoupled and that inhibition of TG transfer activity in zebrafish does not result in steatosis and these fish grow normally like wild-type fish. We hypothesize that atomic level details about these two lipid transfer domains may pave the way for selective pharmacological inhibition of TG transfer to lower plasma lipids without causing the adverse effects of cellular lipid retention. The fundamental question we are asking is: “how MTP distinguishes different lipid ligands and what are the consequences of inhibiting TG transfer activity?” Aim 1: Characterize the different lipid-binding sites in MTP: We will solve MTP structures with different lipid ligands to obtain atomic level details. Mutational analysis will elucidate amino acid residues critical for binding of specific lipids. Aim 2: Identify conformational changes in MTP and PDI subunits during lipid transfer, and different PDI family members that interact with MTP subunit: We hypothesize that conformational changes in both the MTP and PDI subunits occur to accommodate different lipids. We will perform site-directed mutagenesis in the flexible loop region of MTP and a’ domain of PDI to dissect out the mechanisms for this. Although PDI is obligatory for MTP activity, the specificity of different PDI paralogs is unknown. We will test the hypothesis that other PDI family members interact with the MTP subunit and these interactions have physiological consequences. Aim 3: Assess the biological consequences of abolishing TG transfer activity of MTP: After identifying further mutations that abolish TG transfer, we will determine whether these mutants support apoB secretion in cells, lower plasma lipids in mice, and sustain normal fish growth. The proposed studies will provide novel information about 1) the domains and amino acids in the transfer of different lipids by MTP; 2) conformational changes that occur during transfer of different lipids; and 3) biochemical, physiological and organismal consequences of mutating these critical residues. This new knowledge will be invaluable, in the future, to develop novel and TG transfer specific inhibitors of MTP.