title: Highly site-selective stability increases by glycosylation of dihydrofolate reductase.
abstract: Post-translational glycosylation is one of the most abundant forms of covalent protein modification in eukaryotic cells. It plays an important role in determining the properties of proteins, and stabilizes many proteins against thermal denaturation. Protein glycosylation may establish a surface microenvironment that resembles that of unglycosylated proteins in concentrated solutions of sugars and other polyols. We have used site-directed mutagenesis to introduce a series of unique cysteine residues into a cysteine-free double mutant (DM, C85A/C152S) of dihydrofolate reductase from Escherichia coli (EcDHFR). The resulting triple mutants, DM-N18C, DM-R52C, DM-D87C and DM-D132C EcDHFR, were alkylated with glucose, N-acetylglucosamine, lactose and maltotriose iodoacetamides. We found little effect on catalysis or stability in three cases. However, when DM-D87C EcDHFR is glycosylated, stability is increased by between 1.5 and 2.6 kcal.mol(-1) in a sugar-dependent manner. D87 is found in a hinge region of EcDHFR that loses structure early in the thermal denaturation process, whereas the other glycosylation sites are found in regions involved in the later stages of temperature-induced unfolding. Glycosylation at this site may improve the stability of EcDHFR by protecting a region of the enzyme that is particularly prone to denaturation.
authors.affiliation: Cardiff University, UK.
authors.forename: Lai-Hock
authors.initials: LH
authors.lastname: Tey
authors.forename: E Joel
authors.initials: EJ
authors.lastname: Loveridge
authors.forename: Richard S
authors.initials: RS
authors.lastname: Swanwick
authors.forename: Sabine L
authors.initials: SL
authors.lastname: Flitsch
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D055162:Biocatalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006031:Glycosylation; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056811:Unfolded Protein Response
chemical_list: D008024:Ligands; D013762:Tetrahydrofolate Dehydrogenase