Abstract. Integrins are cell surface receptors that mediate numerous interactions between cells and their environment. Binding of integrin α5β1 to its ligand fibronectin in the extracellular matrix plays fundamental roles in cellular adhesion and differentiation. In blood cells these interactions mediate biological processes including erythropoiesis and monocyte adhesion and contribute to pathologies such as sickle cell disease, dysregulation of hematopoiesis, atherosclerosis, and fibrosis. Integrin α5β1 undergoes two distinct conformational changes: extension at the ‘knees’ and opening of the ligand-binding headpiece. These changes give rise to an ensemble of three interconverting integrin conformational states on cell surfaces: low-affinity bent-closed (BC) and extended-closed (EC) conformations and a high-affinity extended-open (EO) conformation. This proposal leverages ground-breaking work under the previous award in which we measured free energy and intrinsic affinity of each integrin α5β1 state. Using the same Fab tools as used for these equilibrium measurements to stabilize the extended, closed, or open α5β1 conformations, we now propose three aims. In Aim 1, we explore how Mn2+, high Mg2+, and low Ca2+ concentrations activate integrins. Our preliminary results show that Mn2+ and high Mg2+ both increase the population of the EO state and increase its intrinsic affinity for ligand and that these effects are dependent on the ADMIDAS metal-ion binding site. To examine why cell surface α5β1 is so stable in the BC state, we test the hypothesis that the α and β-subunit TM domains separate from one another in both the EC and EO states. Aims 2 and 3 measure kinetics to map the activation trajectory of integrin α5β1, i.e. the sequence of ligand binding and conformational change events that occur between the resting state, when 99.8% of unliganded integrin α5β1 is in the BC state, and the final, functional liganded EO state (EO•L) state that is bound to fibronectin and is stabilized by tensile force that is applied to the integrin by actin retrograde flow and resisted by fibronectin in the matrix. In Aim 2, we measure the intrinsic ligand-binding kinetics of each state (kon and koff). Our preliminary data indicates, surprisingly, that the low-affinity BC and EC states bind more rapidly to ligand than the EO state, which is compensated by the >10,000-fold slower off-rate of the EO state. In Aim 3, we measure the kinetics of integrin conformational change using single-molecule FRET probes that measure either the extension or opening steps in the presence or absence of conformation- specific Fabs and ligand. Kinetics of all transitions between the BC, EC, and EO states for unliganded and ligand-bound single integrin molecules will be determined for both purified, soluble ectodomain and intact integrins on blood cells using TIRF microscopy with high temporal resolution. We expect to show an integrin activation trajectory in which ligand binds to the BC+...