The focus of the proposed research is on structural plasticity and large-scale dynamics as determinants of protein function. Detailed and highly specific characterization of functionally important transient non-native protein states will rely on development of experimental strategies combining hydrogen/deuterium exchange in solution (HDX), electrospray ipnization (ESI MS) and gas phase protein ion fragmentation. The new strategies will provide conformer-specific structural and dynamic information by using HDX in combination with (i) simultaneous fragmentation of several ionic species representing a single protein state and (ii) covalent trapping of native and non-native protein states. A new method to probe stability of protein cores by monitoring HDX selectively at polar side chains will also be developed. Multi-stage protein ion fragmentation in both positive and negative ion modes in combination with solution-phase HDX will be evaluated as a means of characterizing local dynamics within large (>30 kDa) proteins with multiple disulfide bonds. These new strategies will be used to further our understanding of transient disorder in cellular retinoic acid binding proteins I and II (CRABP I and II) as a critical factor controlling binding and release of retinoic acid (RA). Structural characterization of non-native protein states through which ligand binding occurs will be completed, followed by investigation of the dynamic events within CRABP II induced by its binding to a retinoic acid receptor (RAR), which facilitate ligand transfer from the transport protein to the receptor. The new experimental strategies will also be applied to further our understanding of metal delivery to cells via the transferrin cycle by examining dynamics and metal-binding properties of serum transferrin (Tf) and their modulation by transferrin receptor (TfR). Understanding the molecular mechanisms of RAR/CRABP and Tf/TfR interactions will be invaluable for the enhancing of a variety of therapeutic strategies exploiting the pleiotropic properties of retinoic acid and the ability of Tf to deliver cytotoxic ligands to malignant cells.