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Coiled-coil fusions are a useful approach to enforce dimerization in protein engineering. However, the final structures of coiled-coil fusion proteins have received relatively little attention. Here, we determine the structural outcome of adjacent parallel and antiparallel coiled coils. The targets are coiled coils that stabilize myosin-10 in single-molecule biophysical studies. We reveal the solution structure of a short, antiparallel, myosin-10 coiled-coil fused to the parallel GCN4-p1 coiled coil. Surprisingly, this structure is a continuous, antiparallel coiled coil where GCN4-p1 pairs with myosin-10 rather than itself. We also show that longer myosin-10 segments in these parallel/antiparallel fusions are dynamic and do not fold cooperatively. Our data resolve conflicting results on myosin-10 selection of actin filament bundles, demonstrating the importance of understanding coiled-coil orientation and stability.

Adipocyte fatty acid binding protein (FABP4) is a 132-aa intracellular lipid binding protein involved in the transport of fatty acids between cell membranes and organelles. FABP4 participates in several pathways including lipolysis and lipogenesis, and is involved in lipid and energy metabolism related diseases such as diabetes. Additionally, in animal models, inhibitors of FABP4 have been found to halt the progression of diabetes that is usually concurrent with obesity. Although the structure of FABP4 has been determined using x-ray crystallography and binding to several of its hydrophobic ligands well characterized, the transitions in the structural dynamics upon ligand binding have yet to be investigated. Here, we report the results of the NMR resonance assignment of the apo form of human FABP4, and compare these results to chemical shift prediction analysis performed using SHIFTX and SPARTA. We also report spin relaxation measurements used to probe the fast (ps-ns) backbone dynamics. Finally, through NMR titrations, we are investigating the structural dynamics transitions which occur upon binding of FABP4 to its hydrophobic ligands, including several novel lipid ligands. The project is a working collaboration between St. Catherine University, the University of Minnesota, and the Minnesota NMR Center, and provides a model of conducting collaborative undergraduate research in partnership between a PUI, a major research institution, and an instrument center.

AFABP is a 132-aa intracellular lipid binding protein involved in the transport of fatty acids between cell membranes and organelles. AFABP participates in several pathways including lipolysis and lipogenesis, and strongly impacts lipid and energy metabolism related diseases such as diabetes. Although the structure of AFABP has been determined using x-ray crystallography and binding to several of its hydrophobic ligands have been characterized, the transitions in the structural dynamics upon ligand binding to leukotrienes have yet to be investigated. Disruption of AFABP reduces inflammation and protects against obesity-induced insulin resistance in animal models, however the exact mechanism of this protective effect is unknown. It is known that FABPs increase the half-life of unstable epoxide-containing LTA4 (the precursor to LTB4 and LTC4), and suggests that this stability in LTA4 against hydrolysis may result in increased inflammatory signaling. It is possible that there may be a subsequent stabilizing interaction between AFABP and LTB4. Elucidating the structural mechanism by which AFABP binds to these eicosanoids is critical to understanding the relationship between AFABP and inflammatory response in macrophages and adipocytes. In this study, apo-AFABP and holo-AFABP bound with LTB4 are studied using solution-state NMR techniques to see how conformational changes in the barrel contribute to the ligand selection mechanism of AFABP. The project is a working collaboration between St. Catherine University, the University of Minnesota, and the Minnesota NMR Center, and provides a model of conducting collaborative undergraduate research in partnership between a PUI, a major research institution, and an instrument center.