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Probing protein small ligand binding dynamics by hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Hui Xiao, University of Massachusetts Amherst


Cellular Retinoic acid binding protein I (CRABP I), a member of intracellular lipid binding proteins (iLBPs), binds physiologically to a mostly hydrophobic ligand, all-trans Retinoic Acid (RA). The binding site is inside an internal cavity, inaccessible in a static apo-protein conformation. Binding of RA to CRABP I does not result in significant changes of the protein tertiary structure, suggesting significance of dynamics in the ligand recognition and binding processes. One of the proposed scenarios for the protein-ligand binding process invokes the notion of a flexible portal region adjacent to the binding site, while another model suggests that the requisite dynamic events are induced by dimerization of the apo-protein in solution. In this work RA binding to CRABP I is studied in dilute solutions (low micro-molar range), where no dimer and/or oligomer formation occurs. Modulation of backbone dynamics within various segments of the protein by its ligand is assessed using a combination of hydrogen exchange, electrospray ionization mass spectrometry and collision-induced dissociation of protein ions in the gas phase. Consistent with the portal model of ligand entry, several protein segments are flexible in the absence of the ligand. At the same time, the two segments containing arginine residues forming a salt bridge with RA form the least flexible region in the apo-form of the protein. Although the presence of RA in solution reduces flexibility of all protein segments, the largest effect is observed within four strands that form one of the two β-sheets enveloping a cavity, which houses the ligand-binding site. These results are consistent with a model in which ligand binding occurs through a partially unstructured state of the protein with unobstructed access to the ligand-binding site. This intermediate (whose core is formed by the two stable arginine-containing strands) corresponds to a relatively low-energy local minimum on the apo-protein energy surface and is frequently sampled under native conditions. Intermediate states were visualized under various mildly denaturing conditions and characterized by HDX/CAD ESI MS. The similar amide backbone protection pattern suggests that the intermediate states bear significant resemblance with the activated intermediate state.

Subject Area

Analytical chemistry

Recommended Citation

Xiao, Hui, "Probing protein small ligand binding dynamics by hydrogen/deuterium exchange and electrospray ionization mass spectrometry" (2005). Doctoral Dissertations Available from Proquest. AAI3163719.