Thermal Stability Assay for Glaucoma‐Causing Myocilin Mutants using Maltose Binding Fusion Protein (MBP)

Abstract

Myocilin is a protein closely linked to inherited forms of glaucoma, a neurodegenerative disorder associated with increased intraocular pressure. In myocilin, over 90% of reported glaucoma‐causing missense mutations are localized to the C‐terminal olfactomedin (OLF) domain. To determine the relative stability of wild‐type and mutant OLF, we developed a fluorescence thermal stability assay without removal of MBP. The fluorescence thermal shift assay can accommodate relatively low concentrations of protein (0.25–0.5 mg/ml) and uses a real time PCR (RT‐PCR) instrument to conduct a slow melt while measuring fluorescence. The excitation and emission settings of the RT‐PCR instrument are compatible with Sypro Orange, a dye known to bind to hydrophobic regions of proteins, which become increasingly exposed as the temperature of the protein‐dye solution is raised. Given upper and lower limits of the fluorescence signal, a melting point can be determined as the midpoint of the unfolding transition. We expressed and purified the OLF (amino acids 228–504) domain of myocilin and select glaucoma mutants (D380A, I477N, I477S, K423E) as maltose‐binding fusion (MBP) proteins in E. coli. In comparison to wild‐type, all four MBP‐OLF mutants tested were destabilized but retained a discrete melting transition expected for a folded protein. Our study appears to be a novel application of the fluorescence stability assay to a MBP fusion protein and lays the foundation for the identification of tailored therapeutic molecules to delay the onset of glaucoma. This work was supported by NSF REU #0851780, Glaucoma Research Foundation (Shaffer Fund), and American Health Assistance Foundation.