tailieunhanh - Báo cáo khoa học: Highly site-selective stability increases by glycosylation of dihydrofolate reductase

Post-translational glycosylation is one of the most abundant forms of cova-lent 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 micro-environment that resembles that of unglycosylated proteins in concentrated solutions of sugars and other polyols. | ỊFEBS Journal Highly site-selective stability increases by glycosylation of dihydrofolate reductase Lai-HockTey1 E. Joel Loveridge1 Richard S. Swanwick1 Sabine L. Flitsch2 and Rudolf K. Allemann1 1 Schoolof Chemistry Cardiff University UK 2 Schoolof Chemistry and Manchester Interdisciplinary Biocentre University of Manchester UK Keywords enzyme glycosylation kinetics mutagenesis stability Correspondence R. K. Allemann Schoolof Chemistry Cardiff University Main Building Park Place Cardiff CF10 3AT UK Fax 44 29 2087 4030 Tel 44 29 2087 9014 E-mail allemannrk@ Present address Department of Life Sciences Imperial College London UK Received 10 January 2010 revised 26 February 2010 accepted 2 March 2010 doi 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 and 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

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