Spectroscopic Studies of Glycosylated CEACAM1 Ectodomains and Membrane Protein Folding in Lipid-Detergent Mixtures

Biomolecular nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy are established methods for determining the structure and dynamics of macromolecules. The work in this dissertation utilized magnetic resonance and SAXS methods to investigate two major research aims that are outlined in the following sections.

The first study describes the human carcinoembryonic antigen-like cellular adhesion molecule 1 (CEACAM1), which facilitates cell-cell adhesion in healthy tissue. Impaired CEACAM1-mediated cellular adhesions result in tumor formation, and CEACAM1 is also targeted by surface proteins of pathogenic bacteria. CEACAM1 is localized to the plasma membrane and contains four highly-glycosylated ectodomains, of which the N-terminal domain is both essential for normal function and specific to pathogen interactions. Investigating the CEACAM1 N-domain is therefore of broad interest for relevant disease states. However, the majority of studies with isolated CEACAM1 N-domain proteins lack glycan modifications that may be important for CEACAM1 homotypic and heterotypic interactions. In this work, EPR spectroscopy was used to investigate four CEACAM1 N-domain glycoforms. All CEACAM1 N-domain proteins form a homodimer in solution, and, contrary to previous literature, glycosylation does not perturb CEACAM1 dimerization. Furthermore, the interaction interface between CEACAM1 N-domains is maintained across all glycoforms. Collectively, glycosylated CEACAM1 N-domain proteins recapitulate native homotypic interactions, and these results provide a foundation for understanding the role of CEACAM1 glycans in vivo.

The second major aim of this dissertation investigated a subclass of membrane mimics used to solubilize membrane proteins. Approximately 50% of drugs on the market target membrane proteins, and they are often isolated outside of the native membrane for structural and functional studies. Membrane proteins are commonly solubilized in detergent micelles, but lipid-detergent mixtures, or bicelles, represent another water-soluble amphipathic assembly. The ideal bicelle morphology contains a phase-separated detergent rim and lipid core, which is meant to provide a bilayer-like environment for an embedded membrane protein. This work described the morphologies of eleven lipid-detergent compositions without protein using SAXS. For each investigated mixture, evidence of internal lipid organization occurred when the ratio of lipid to detergent was at least 1:2. Results suggest that bicelles undergo a composition-independent phase transition that requires a minimum number of lipids, and the detergent component modulates bicelle size. To assess the impact of bicelle phase transitions and size in a system containing protein, a model, polytopic α-helical transmembrane protein was prepared in bicelles characterized with SAXS. The overall protein fold and tertiary helical contacts, evaluated with NMR and EPR spectroscopy, demonstrated that bicelles stabilize the protein fold in a unique manner from detergent micelles. These results suggest that bicelle lipid and detergent molecules may rearrange to stabilize protein folds through preferential lipid solvation and/or alleviating hydrophobic mismatch.