Highly site-selective stability increases by glycosylation of dihydrofolate reductase.
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.
Tey
Lai-Hock
LH
Cardiff University, UK.
Loveridge
E Joel
EJ
Swanwick
Richard S
RS
Flitsch
Sabine L
SL
Allemann
Rudolf K
RK
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
D008024:Ligands; D013762:Tetrahydrofolate Dehydrogenase