Toward the dynamic structure of the Escherichia coli integral membrane protein glycerol facilitator
| dc.contributor.author | Hernando, Mary D. | |
| dc.contributor.examiningcommittee | Court, Deborah (Microbiology) | en_US |
| dc.contributor.examiningcommittee | Stetefeld, Jorg (Chemistry) | en_US |
| dc.contributor.examiningcommittee | Khajehpour, Mazdak (Chemistry) | en_US |
| dc.contributor.examiningcommittee | Rainey, Jan (Biochemistry and Molecular Biology, Dalhousie University) | en_US |
| dc.contributor.supervisor | O'Neil, Joe (Chemistry) | en_US |
| dc.date.accessioned | 2020-01-06T20:18:27Z | |
| dc.date.available | 2020-01-06T20:18:27Z | |
| dc.date.issued | 2019-12-20 | en_US |
| dc.date.submitted | 2019-12-30T02:38:42Z | en |
| dc.degree.discipline | Chemistry | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.abstract | The aquaporin family of integral membrane proteins (IMPs) are found in all forms of life, from prokaryotes to humans. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Though the structure of GF was solved in 2000, little is known about the dynamics of GF and the role the dynamics play in the function and stability of the protein. This lack of information pertaining to dynamics is common for many IMPs. Here, preparations of isotope-labelled GF for solution and solid-state nuclear magnetic resonance (NMR) spectroscopy are explored and optimized in order to reveal atomic dynamics of the protein. The stability of the GF homotetramer solubilized in various agents, including detergents, bicelles, lipid nanodiscs, random heteropolymers, and other buffer additives is explored. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), intrinsic tryptophan fluorescence, size exclusion chromatography multi-angle light scattering (SEC-MALS), dynamic light scattering (DLS), and differential scanning calorimetry (DSC) were used to determine the oligomeric forms of GF and probe its stability. Using these techniques, it was found that GF tetramers self-associate to form octamers and higher Mr oligomers 24 hours after solubilization. The protein was found to be most stable in lauryl maltose neopentyl glycol (LMNG), where it existed in a tetramer-octamer equilibrium for 9 days. The structures of the GF tetramer and octamer were explored by negative stain electron microscopy (EM), size-exclusion chromatography small-angle light scattering (SEC-SAXS), and solid-state magic-angle spinning (MAS) NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure determination, negative stain EM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer. The octamer forms from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. | en_US |
| dc.description.note | February 2020 | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/34448 | |
| dc.language.iso | eng | en_US |
| dc.rights | open access | en_US |
| dc.subject | glycerol facilitator | en_US |
| dc.subject | membrane protein | en_US |
| dc.subject | negative stain transmission electron microscopy | en_US |
| dc.subject | size-exclusion chromatography multi-angle light scattering | en_US |
| dc.subject | small angle x-ray scattering | en_US |
| dc.subject | solid-state NMR spectroscopy | en_US |
| dc.title | Toward the dynamic structure of the Escherichia coli integral membrane protein glycerol facilitator | en_US |
| dc.type | doctoral thesis | en_US |