| Gangliosides are found in high concentrations in the plasma membranes of neuronal cells. Their catabolism, a vital process for normal cell function, is carried out by sequential cleavage of sugar groups by exohydrolases that require accessory proteins for activity. The GM2 Activator Protein (GM2AP) specifically recognizes ganglioside GM2 and presents the oligosaccharide group to beta-hexosaminidase A (Hex A) for cleavage. Mutations in Hex A and GM2AP lead to a build up of GM2, causing neurodegenerative diseases such as Tay Sachs disease or AB variant gangliosidosis.;This work utilized site directed spin labeling (SDSL) and electron paramagnetic spectroscopy (EPR) to determine the membrane bound orientation of GM2AP on lipid bilayers and to investigate conformational changes of mobile loops. A paramagnetic nitroxide radical, (1-oxyl-2,2,5,5,-tetramethyl-Delta 3-pyrroline-3-methyl) methanethiosulfonate (MTSL), was attached to GM2AP via disulfide bond to a non-native cysteine residue. Continuous wave power saturation experiments were used for measuring changes in the EPR relaxation properties of spin labeled proteins in the presence of a surface bound paramagnetic collider, DOGS-NTA-Ni, to determine a membrane bound orientation of GM2AP on phosphatidylcholine bilayers. Collision values with DOGS-NTA-Ni were used to map regions of the protein that were accessible and inaccessible to the surface bound collider. Sites of high collision (T90R1, L126R1, and N136R1) were interpreted to be located near the bilayer surface, which corresponded to an orientation where the face of the lipid binding cavity lies on the bilayer surface.;Conformational changes of two flexible loops (spanning residues 58-62 and 122-136) were also investigated using SDSL EPR. Continuous wave EPR spectra for six spin labeled sites in these two loops in the presence and absence of GM2 micelles did not reflect changes in mobility associated with a conformational change upon ligand binding. Including the six loop sites, solution EPR spectra of four additional spin labeled sites were obtained and fit using the microscopic order macroscopic disorder (MOMD) model. The six spectra from the flexible loop sites required two components to obtain a best fit. Temperature studies suggested that the 122-136 loop was in conformational exchange, but the 58-62 loop showed no temperature dependent correlation to protein dynamics. |
