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

Highly site-selective stability increases by glycosylation of dihydrofolate reductase Lai-Hock Tey 1 , E. Joel Loveridge 1 , Richard S. Swanwick 1, *, Sabine L. Flitsch 2 and Rudolf K. Allemann 1 1 School of Chemistry, Cardiff University, UK 2 School of Chemistry and Manchester Interdisciplinary Biocentre, University of Manchester, UK Introduction Post-translational glycosylation is one of the most abundant forms of covalent protein modification in eukaryotic cells and plays an important role in deter- mining the properties of proteins, affecting many molecular processes in vivo [1–5]. There are two main types of protein glycosylation: N-glycosylation, in which the oligosaccharide is attached to an asparagine side chain, and O-glycosylation, in which it is attached to the side chain of serine or threonine residues [4]. Surface glycoproteins act as markers for inter- and intracellular communication, and glycosylation has been shown to affect a number of protein properties such as structure, dynamics, stability and catalytic activity [6–14]. Glycosylation stabilizes many proteins against ther- mal denaturation [14–17], whereas the removal of car- bohydrates from naturally glycosylated proteins can lead to decreased thermal stability and an increased tendency towards protein aggregation [18–20]. Some studies have shown that glycans reduce the rate of unfolding but do not affect refolding of denatured pro- teins, leading to the conclusion that glycans preferen- tially bind to the folded protein and therefore stabilize it [20–24]. Others have also shown that folding is pro- moted in the presence of glycans [18,25,26], suggesting that the effects are protein specific. Notably, many proteins show considerable increases in thermostability when in solution with high concentrations of sugars or Keywords enzyme; glycosylation; kinetics; mutagenesis; stability Correspondence R. K. Allemann, School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK Fax: +44 29 2087 4030 Tel: +44 29 2087 9014 E-mail: allemannrk@cf.ac.uk *Present address Department of Life Sciences, Imperial College, London, UK (Received 10 January 2010, revised 26 February 2010, accepted 2 March 2010) doi:10.1111/j.1742-4658.2010.07634.x 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. We have used site-directed mutagen- esis 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 kcalÆmol )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. Abbreviations DM, double mutant; EcDHFR, Escherichia coli dihydrofolate reductase. FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS 2171 other polyols [27]. Protein glycosylation may therefore establish a surface microenvironment that resembles that of unglycosylated proteins in such solutions. Several methods have been described for the genera- tion of neoglycoproteins via site-selective glycosylation of proteins using chemical modification of biotechno- logically produced proteins [2,3,28–31]. One such approach combines site-directed mutagenesis, to intro- duce unique cysteine residues at the required sites, and a highly flexible but selective chemical derivatization strategy (Scheme 1) in which reaction of the free thiol group of a cysteine residue with a synthetic glycosyl iodoacetamide produces a stable linkage between the protein and the carbohydrate [30] which resembles that found in native glycosylation of asparagines [32–34]. We have previously used this approach to study of the effect of site-specific glycosylation on the physical and chemical properties of the naturally nonglycosylated Scheme 1. Strategy used for the synthesis of highly purified glycosylated Escherichia coli dihydrofolate reductase triple mutants [30]. A unique cysteine residue on the pro- tein is first reacted with a glycosyl iodoace- tamide (glucose is used as an example here); unalkylated proteins are biotinylated by reaction with 2-((biotinoyl)amino)ethyl methanethiosulfonate. Treatment with resin-bound avidin removes the biotinylated protein from solution, leaving highly purified neoglycoprotein. Glycosylation of E. coli DHFR L H. Tey et al. 2172 FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS enzyme dihydrofolate reductase (5,6,7,8-tetrahydro- folate : NADP + oxidoreductase, EC 1.5.1.3) from Escherichia coli (EcDHFR) [14]. EcDHFR catalyses the stereospecific reduction of 7,8-dihydrofolate to (6S)-5,6,7,8-tetrahydrofolate using NADPH as a cofac- tor [35], and is therefore responsible for maintaining the tetrahydrofolate pool within the cell. EcDHFR is a monomeric enzyme made up of eight b-strands, four a helices and a number of important loop regions; it is typically divided into three subdomains, the adenosine- binding domain, the substrate-binding domain and the loop domain (Fig. 1) [36]. Our previous study was based on a cysteine-free C85A ⁄ C152S double mutant of EcDHFR (DM EcDHFR), which has similar fold- ing, stability and kinetic properties to the wild-type enzyme (WT EcDHFR) [37]. Cysteine residues were introduced at two sites and the effect of glycosylation at these sites was studied [14]. Substitution of a cysteine residue at position 87 (to form DM-D87C EcDHFR) caused a loss in thermostability of the protein that was reversed on glycosylation, whereas DM-E120C EcDHFR had similar thermostability to the native enzyme and subsequent glycosylation led to a smaller increase in melting temperature than that observed at position 87 [14]. The kinetic parameters of the steady-state reaction catalysed by EcDHFR were not significantly affected by mutation and subsequent glycosylation at either position [14]. This difference in response to glycosylation at the two sites was intrigu- ing and prompted further study. Here, we describe the effect of glycosylation at three further sites on EcDHFR and report the kinetic properties, thermal stability and chemical stability at room temperature of the resulting glycoproteins. The sites chosen were N18, on the catalytically important M20 loop, R52, respon- sible for binding the glutamate tail of the substrate, and D132, ‘behind’ the active site at the end of the FG loop (Fig. 1). Our results suggest that the local envi- ronment of the protein is critically important in deter- mining the effect of the glycosyl chain on protein unfolding. Results Preparation of glycosylated EcDHFR mutants Double and triple mutants of EcDHFR were prepared using standard molecular biology techniques and the proteins expressed, purified, glycosylated and further purified as described previously [30]. Prior to glycosyl- ation, all proteins were > 95% pure as judged by SDS–PAGE. Glycosylation was confirmed by tryptic digestion followed by MALDI-TOF MS (Fig. S1). Ligand binding and kinetics of glycosylated EcDHFR Quenching of the enzyme fluorescence at 340 nm was used to determine the equilibrium dissociation con- stants of enzyme–NADPH and enzyme–folate com- plexes. All five mutants have K D values similar to WT EcDHFR for both NADPH and folate (supporting information). The largest change was seen for DM-R52C with folate, where a threefold loss of affin- ity was seen. In addition, no significant differences between the kinetic parameters of the five mutants and those of the wild-type protein were observed in either the steady state or pre-steady state, nor were there any reliable trends in the values on glycosylation (support- ing information). Stability of glycosylated EcDHFR The far-UV CD spectra of the EcDHFR double and triple mutants and of the glycosylated triple mutants Fig. 1. Structure of Escherichia coli dihydrofolate reductase (PBD 1RA2) [36] showing the position of the five residues mutated to cysteine for this study. The two views are rotated 180° about the z-axis relative to each other. The adenosine-binding domain (ABD), substrate-binding domain (SBD), loop domain (LD) and spe- cific loops mentioned in the discussion are indicated. The enzyme is shown as a cartoon representation; residues of interest and ligands are shown as sticks. H 2 F, 7,8-dihydrofolate. L H. Tey et al. Glycosylation of E. coli DHFR FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS 2173 were all similar to those of the wild-type enzyme, indi- cating that neither the mutations per se nor glycosyla- tion had an effect on the secondary structure of the proteins large enough to be detectable by CD spectros- copy (supporting information). Thermal denaturation of WT EcDHFR and the five mutants was reversible from 80 to 20 °C, and the melting temperatures of all proteins except DM-D87C EcDHFR were similar (Table 1). It has previously been shown that DM EcDHFR has similar stability to the WT protein [37]. As previously reported, the thermal denaturation tem- perature of DM-D87C EcDHFR is almost 10 °C lower than that of WT EcDHFR, even though there is no significant difference in the secondary structure of the two proteins, and stability is restored by glycosylation [14]. The change in stability is because of the glycan rather than the acetamide linkage [14]. Stability of the glycosylated mutant proteins was also determined using equilibrium urea titrations monitored by trypto- phan fluorescence emission (Table 2 and supporting information). Mirroring the thermal stability results, DM EcDHFR and three of the four triple mutants showed little change in resistance to urea denaturation, although DM-D87C EcDHFR showed a considerably lower free energy of unfolding, indicating a signifi- cantly lower stability. The free energy of unfolding of DM-D87C EcDHFR was increased by glycosylation, although the other mutants were unaffected. The sta- bility of glycosylated DM-D87C EcDHFRs increased with the length of the glycosyl chain; monosaccharides caused a similar increase in free energy of unfolding as incubating the nonglycosylated enzyme in a 0.5 m solu- tion of maltose, whereas larger sugars gave a more pronounced effect. Discussion We have previously reported a large reduction in ther- mal stability for DM-D87C EcDHFR and its subse- quent ‘rescue’ by glycosylation [14]. The same study showed a slight increase in thermal stability on glyco- sylation of DM-E120C EcDHFR. Here we demon- strate that three further EcDHFR triple mutants show similar stability (against both temperature- and urea- induced denaturation) to the wild-type protein and that, in these cases, glycosylation does not improve Table 1. Melting temperatures of EcDHFR, its mutants and their glycosylated forms. Values were determined by CD spectroscopy using 10 l M enzyme in 5 mM potassium phosphate buffer (pH 7.0). DM, double mutant; EcDHFR, Escherichia coli dihydrofolate reductase; WT, wild-type, ND, not determined. Glycan T m (°C) WT EcDHFR-C85A ⁄ C152S (DM) DM-N18C DM-R52C DM-D87C [14] DM-E120C [14] DM-D132C None 50.7 ± 0.2 50.9 ± 0.9 50.5 ± 0.4 49.7 ± 0.5 40.9 ± 0.3 50.8 ± 0.4 50.7 ± 0.3 Glucose 50.5 ± 0.3 49.9 ± 0.2 47.1 ± 0.3 51.8 ± 0.2 52.8 ± 0.9 N-acetylglucosamine 51.0 ± 0.1 50.2 ± 0.9 49.6 ± 1.1 52.5 ± 0.1 52.1 ± 1.6 Lactose 50.6 ± 0.9 51.6 ± 0.1 46.8 ± 2.1 54.1 ± 0.1 51.2 ± 0.7 Maltotriose 51.0 ± 0.7 49.8 ± 1.0 47.5 a ND 52.4 ± 0.6 0.5 M Maltose 52.9 ± 0.4 53.5 a 53.5 a 43.4 ± 0.4 ND 54.6 a a Single measurement. Table 2. Free energy of unfolding of Escherichia coli dihydrofolate (EcDHFR), its mutants and their glycosylated forms. Values were deter- mined by fluorescence intensity measurement of urea-induced unfolding of 2 l M enzyme in 10 mM potassium phosphate buffer (pH 7.0). DM, double mutant; WT, wild-type. Glycan DG° (kcalÆmol )1 ) WT EcDHFR-C85A ⁄ C152S (DM) DM-N18C DM-R52C DM-D87C DM-D132C None 5.9 ± 0.3 5.2 ± 0.3 5.3 ± 0.3 5.7 ± 0.2 2.7 ± 0.2 5.5 ± 0.3 Glucose 5.7 ± 0.1 5.5 ± 0.2 4.2 ± 0.1 5.3 ± 0.2 N-acetylglucosamine 5.5 ± 0.2 5.4 ± 0.2 4.3 ± 0.3 5.2 ± 0.3 Lactose 5.6 ± 0.3 5.5 ± 0.2 5.1 ± 0.1 5.4 ± 0.3 Maltotriose 5.3 ± 0.3 5.4 ± 0.1 5.3 ± 0.3 5.1 ± 0.2 0.5 M Maltose 6.1 ± 0.2 5.5 ± 0.2 5.4 ± 0.2 4.1 ± 0.1 5.5 ± 0.1 Glycosylation of E. coli DHFR L H. Tey et al. 2174 FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS stability (Table 1). In all cases except that of DM-D87C EcDHFR, site-selective glycosylation had a smaller effect on the thermal stability of the proteins than the presence of 0.5 m maltose. A similar trend was observed for the stabilities of the proteins at ambient temperature with respect to denaturation induced by urea (Table 2). Notably, although the free energy of unfolding of DM-D87C increased with increasing glycan length, the thermal stability did not show such a trend and was instead highest with the monosaccharide N-acetylglucosamine. Glycosylation of DM-D87C EcDHFR with lactose or maltotriose acetamides had a larger effect on the free energy of unfolding than a 0.5 m solution of maltose. Interest- ingly, the changes in free energy of unfolding were because of changes in the gradient of the urea depen- dence of the free energy (supporting information), rather than changes to the mid-point of the urea- induced unfolding. This suggests that glycosylation of DM-D87C EcDHFR affects the cooperativity of the unfolding transition rather than simply the resistance to denaturants [38]. The results presented here provide further support [14] that, at least in the case of dihy- drofolate reductase, increased stability through the addition of glycans is because of highly site-specific effects rather than nonspecific changes to the solvation properties of the enzyme, suggesting that stabilization of EcDHFR relies on specific interactions between the protein and the glycan. In the case of DM-D87C EcDHFR, it appears that glycosylation increases the effective concentration of sugar at a critical site to more than that provided by a 0.5 m solution of malt- ose. In fact, the increase in melting temperature is sim- ilar to that seen in a 1.5 m (50% w⁄ v) solution of sucrose [39]. By contrast, site-selective glycosylation of the protein in regions unimportant for glycan-induced stability would produce no benefit, as observed here. Inspection of the EcDHFR structure reveals no fea- ture around position 87 that would be expected to interact particularly favourably with glycans. Compu- tational [40,41] and experimental [42] work has indi- cated that the ‘hinge’ region of the adenosine-binding domain in which D87 is found unfolds very early in the denaturation process, although others [43–45] have suggested an alternative folding pathway in which this region would be expected to unfold slightly later. If this region does lose structure early in the unfolding process, this may explain why the stability of EcDHFR is sensitive to glycosylation at this site – regions more vulnerable to denaturation are likely to benefit more from additional stabilizing interactions. N18, E120 and D132 are all found in the loop domain, which retains structure until relatively late in the thermal unfolding process [40,41], whereas R52 is formally located in the adenosine-binding domain but forms part of the substrate-binding pocket. Sugars bound at position 52 are therefore more likely to interact with the relatively stable [40–42] substrate- binding domain (Fig. 1). Hence glycosylation at these positions may not exert a similarly stabilizing effect as glycosylation at position 87. Both ligand binding and the kinetics of EcDHFR were remarkably robust to the mutations made and subsequent glycosylation. The most notable difference in K D values was observed for DM-R52C EcDHFR with folate, although this is still only an approximately threefold increase. R52 forms part of the binding site for the glutamate tail of the folate ligand, whereas N18 forms part of the M20 loop, which closes over NADPH after it enters the active site (Fig. 1). It has previously been shown that reacting DM-N18C and DM-E17C mutants with bulky groups has little effect on their kinetics or ligand binding relative to EcDHFR [46,47]. E120 and D132 are both located on the FG loop, important because of its interactions with the M20 loop that controls progression through the cata- lytic cycle [36,48]. Mutation of glycine 121 to bulkier residues causes a sharp decrease in catalytic activity, and a reduction in the affinity for NADPH [49,50]. However, this is likely to be because of global struc- tural changes observed for the G121V mutant [40], which disrupt the ability of the EcDHFR : NADPH complex (and the reactive Michaelis complex) to form its native ‘closed’ conformation [48]. Changes at posi- tion 120, where the side chain is exposed to solvent, would not be expected to produce so pronounced an effect. The absence of large effects on catalysis pro- vides further evidence that mutation and subsequent glycosylation do not produce significant changes in the global structure of the enzyme, but that stability of EcDHFR may be affected by binding of sugars to specific sites on the enzyme. In conclusion, our previous study suggested that the thermal stability of proteins can be increased signifi- cantly by the attachment of even relatively small car- bohydrates, rather than the larger oligosaccharides typically found in nature [14]. We now report a similar effect on chemical stability at room temperature, and add that the local environment of the protein appears to be critically important in determining the effect of bound oligosaccharides. The large oligosaccharides observed in nature may allow greater coverage of a number of discrete, critical points of stabilization from a single glycosylation site, rather than being simply caused by blanket coverage of large regions of the protein surface. Alternatively, increases in stability L H. Tey et al. Glycosylation of E. coli DHFR FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS 2175 because of protein–carbohydrate interactions close to the attachment site may be coupled to other functions (such as molecular recognition) at the ends of the gly- can chain. Our results suggest that glycosylation at position 87 of EcDHFR may improve its stability by protecting a region that is particularly prone to denaturation. Experimental procedures Protein preparation EcDHFR triple mutants were generated by site-directed mutagenesis of DNA encoding DM EcDHFR in the same way as for DM-E120C EcDHFR [30]. Mutagenic primers were: 5¢-CGCGTTATCGGCATGGAA TGCGCCATGCC GTGG-3¢ (N18C), 5¢-CTGGGAATCAATCGGT TGCCCG TTGCCAGGAC-3¢ (R52C), 5¢-GCGGCGGCGGGT TGC GTACCAGAAATCATGG-3¢ (D87C) and 5¢-CCGGATTA CGAGCCGGAT TGCTGGGAATCGG-3¢ (D132C). The cysteine codons are underlined. All unglycosylated proteins were purified by methotrexate affinity and anion-exchange chromatography as described previously [40]. Purified triple mutants were subsequently glycosylated with glucose, N-acetylglucosamine, lactose and maltotriose acetamides and further purified as described previously for DM-E120C EcDHFR [30]. CD spectroscopy Experiments were performed using an Applied Photophys- ics (Leatherhead, UK) Chirascan spectrometer at a protein concentration of 10 lm in 5 mm potassium phosphate buf- fer (pH 7.0). Spectra were acquired between 200 and 280 nm. To monitor thermal denaturation, spectra were acquired between 20 and 80 °C using a temperature gradi- ent of 0.4 °CÆmin )1 . Unfolding of the protein was moni- tored at 222 nm and the melting temperature was taken as the midpoint of the observed transition. Thermal denatur- ation measurements were performed in triplicate. Determination of free energy of unfolding Equilibrium unfolding of the proteins and their deriva- tized glycoforms was monitored in the presence of urea by the fluorescence intensity at 345 nm and 20 °C using a Perkin–Elmer (Beaconsfield, UK) LS55 Luminescence spectrometer. Urea solutions were prepared freshly for each experiment and treated with AG Ò 501-X8 ion- exchange resin (Bio-Rad, Hemel Hempstead, UK). The protein concentration was maintained at 2 lm in 10 mm potassium phosphate buffer (pH 7.0) containing 0.1 mm EDTA, 0.1 mm dithiothreitol and the required concentra- tion of urea. All samples were incubated overnight at room temperature prior to measurement. Between 10 and 15 data points were acquired to adequately define the denaturation curve, and the free energy of unfolding was determined using the linear extrapolation method [38]. All unfolding measurements were performed in triplicate. Ligand-binding experiments Equilibrium dissociation constants (K D ) of the protein com- plexes with folate and NADPH were measured at 20 °Cby monitoring quenching of the intrinsic tryptophan fluores- cence as a function of ligand concentration using a Perkin– Elmer LS55 Luminescence spectrometer. Folate was used in place of dihydrofolate because of its enhanced stability. Protein concentrations were 0.05 or 0.5 lm (for titration with NADPH and folate, respectively) in 50 mm potassium phosphate buffer (pH 7.0) containing 50 mm NaCl, 0.1 mm EDTA and 0.1 mm dithiothreitol. Ligand concentrations were 0.1–9.5 lm for NADPH and 1–125 lm for folate. Dissociation constants were determined by fitting the nor- malized fluorescence intensities (F) data to the Langmuir isotherm F Fit ={1+(K D ⁄ [Ligand]) n } )1 , where n = 1 (i.e. 1 : 1 binding) gave the best fits. Enzyme kinetics All kinetic measurements were performed in MTEN buffer (50 mm Mes, 25 mm Tris, 25 mm ethanolamine, 100 mm NaCl pH 7.0) at 20 °C. Steady-state rates were measured spectrophotometrically by following the decrease in absor- bance at 340 nm during the reaction (e 340, NADPH+DHF = 11 800 m )1 Æcm )1 ). The enzyme (10 lm) was incubated with NADPH (20 lm) for 15 min to avoid hysteresis [51]. This enzyme–NADPH solution (5 lL) was added to 950 lL buffer and NADPH (1–100 lm final concentration) added. The reaction was started by adding dihydrofolate (100 lm final concentration). Each experiment was performed in triplicate and the rates calculated from the linear fittings of the initial velocities. K M NADPH and k cat were determined by fitting the data to the Michaelis–Menten equation. The K M value was not determined for dihydrofolate because of the lower stability of this compound. Pre-steady-state kinetic experiments were performed on an Applied Photophysics stopped-flow spectrometer with 2.5 mL drive syringes. EcDHFR (8 lm) was preincubated with NADPH (4 lm) for at least 15 min at 20 °C and the reaction initiated by rapidly mixing with an equal volume of dihydrofolate (100 lm). The dead time of the experiment was < 2 ms. The reaction was monitored by fluorescence energy transfer using a 400 nm cut-off filter and excitation at 292 nm. Rate constants were determined by fitting the observed kinetic traces to single or double exponential decay using software provided with the instrument. Glycosylation of E. coli DHFR L H. Tey et al. 2176 FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS Acknowledgement The financial support from the UK’s Biotechnology and Biological Sciences Research Council (BBSRC) through grants 6 ⁄ B15285 (SLF, RSS and RKA) and BB ⁄ E008380 ⁄ 1 (RKA and EJL) and from Cardiff Uni- versity (studentship to L-HT). References 1 Dwek RA (1996) Glycobiology: toward understanding the function of sugars. Chem Rev 96, 683–720. 2 Gamblin DP, Scanlan EM & Davis BG (2009) Glyco- protein synthesis: an update. Chem Rev 109, 131–163. 3 Davis BG (2002) Synthesis of glycoproteins. Chem Rev 102, 579–601. 4 Spiro RG (2002) Protein glycosylation: nature, distribu- tion, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12, 43R–56R. 5 An HJ, Froehlich JW & Lebrilla CB (2009) Determina- tion of glycosylation sites and site-specific heterogeneity in glycoproteins. Curr Opin Chem Biol 13, 421–426. 6 Rademacher TW, Parekh RB & Dwek RA (1988) Glycobiology. Annu Rev Biochem 57, 785–838. 7 Varki A (1993) Biological roles of oligosaccharides – all of the theories are correct. Glycobiology 3, 97–130. 8 Rudd PM, Joao HC, Coghill E, Fiten P, Saunders MR, Opdenakker G & Dwek RA (1994) Glycoforms modify the dynamic stability and functional-activity of an enzyme. Biochemistry 33, 17–22. 9 Mer G, Hietter H & Lefevre JF (1996) Stabilization of proteins by glycosylation examined by NMR analysis of a fucosylated proteinase inhibitor. Nat Struct Biol 3, 45–53. 10 Kohen A, Jonsson T & Klinman JP (1997) Effects of protein glycosylation on catalysis: changes in hydrogen tunneling and enthalpy of activation in the glucose oxi- dase reaction. Biochemistry 36, 2603–2611. 11 Lowe JB & Marth JD (2003) A genetic approach to mammalian glycan function. Annu Rev Biochem 72, 643–691. 12 Lehle L, Strahl S & Tanner W (2006) Protein glycosyla- tion, conserved from yeast to man: a model organism helps elucidate congenital human diseases. Angew Chem Int Ed 45, 6802–6818. 13 Sola RJ, Rodriguez-Martinez JA & Griebenow K (2007) Modulation of protein biophysical properties by chemical glycosylation: biochemical insights and bio- medical implications. Cell Mol Life Sci 64, 2133–2152. 14 Swanwick RS, Daines AM, Tey LH, Flitsch SL & Allemann RK (2005) Increased thermal stability of site-selectively glycosylated dihydrofolate reductase. ChemBioChem 6, 1338–1340. 15 Baek WO & Vijayalakshmi MA (1997) Effect of chemi- cal glycosylation of Rnase A on the protein stability and surface histidines accessibility in immobilized metal ion affinity electrophoresis (IMAGE) system. Biochim Biophys Acta 1336, 394–402. 16 Masarova J, Mislovicova D, Gemeiner P & Michalkova E (2001) Stability enhancement of Escherichia coli peni- cillin G acylase by glycosylation with yeast mannan. Biotechnol Appl Biochem 34, 127–133. 17 Broersen K, Voragen AGJ, Hamer RJ & de Jongh HHJ (2004) Glycoforms of beta-lactoglobulin with improved thermostability and preserved structural packing. Bio- technol Bioeng 86, 78–87. 18 Wang CQ, Eufemi M, Turano C & Giartosio A (1996) Influence of the carbohydrate moiety on the stability of glycoproteins. Biochemistry 35, 7299–7307. 19 Gimenez-Roqueplo AP, Celerier J, Lucarelli G, Corvol P & Jeunemaitre X (1998) Role of N-glycosylation in human angiotensinogen. J Biol Chem 273, 21232–21238. 20 Kwon KS & Yu MH (1997) Effect of glycosylation on the stability of alpha(1)-antitrypsin toward urea dena- turation and thermal deactivation. Biochim Biophys Acta 1335 , 265–272. 21 Xie GF & Timasheff SN (1997) Mechanism of the sta- bilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured than for the native protein. Protein Sci 6, 211–221. 22 Xie GF & Timasheff SN (1997) The thermodynamic mechanism of protein stabilization by trehalose. Biophys Chem 64, 25–43. 23 Carpenter JF, Crowe LM & Crowe JH (1987) Stabiliza- tion of phosphofructokinase with sugars during freeze- drying – characterization of enhanced protection in the presence of divalent-cations. Biochim Biophys Acta 923, 109–115. 24 Crowe JH, Crowe LM & Chapman D (1984) Preserva- tion of membranes in anhydrobiotic organisms – the role of trehalose. Science 223, 701–703. 25 Kimura N, Uchida M, Nishimura S & Yamaguchi H (1998) Promotion of polypeptide folding by interactions with Asn-glycans. J Biochem 124, 857–862. 26 Sinha S & Surolia A (2007) Attributes of glycosylation in the establishment of the unfolding pathway of soy- bean agglutinin. Biophys J 92, 208–216. 27 Timasheff SN (1993) The control of protein stability and association by weak interactions with water – how do solvents affect these processes. Annu Rev Biophys Biomol Struct 22, 67–97. 28 Bernardes GJL, Castagner B & Seeberger PH (2009) Combined approaches to the synthesis and study of glycoproteins. ACS Chem Biol 4, 703–713. 29 Dondoni A, Massi A, Nanni P & Roda A (2009) A new ligation strategy for peptide and protein glycosyla- tion: photoinduced thiol-ene coupling. Chem Eur J 15, 11444–11449. 30 Swanwick RS, Daines AM, Flitsch SL & Allemann RK (2005) Synthesis of homogenous site-selectively glycosy- lated proteins. Org Biomol Chem 3, 572–574. L H. Tey et al. Glycosylation of E. coli DHFR FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS 2177 31 Watt GM, Lund J, Levens M, Kolli VSK, Jefferis R & Boons GJ (2003) Site-specific glycosylation of an agly- cosylated human IgG1-Fc antibody protein generates neoglycoproteins with enhanced function. Chem Biol 10, 807–814. 32 Macmillan D, Bill RM, Sage KA, Fern D & Flitsch SL (2001) Selective in vitro glycosylation of recombi- nant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin. Chem Biol 8, 133–145. 33 Bill RM, Winter PC, McHale CM, Hodges VM, Elder GE, Caley J, Flitsch SL, Bicknell R & Lappin TRJ (1995) Expression and mutagenesis of recombinant human and murine erythropoietins in Escherichia coli. Biochim Biophys Acta 1261, 35–43. 34 Davis NJ & Flitsch SL (1991) A novel method for the specific glycosylation of proteins. Tetrahedron Lett 32, 6793–6796. 35 Charlton PA, Young DW, Birdsall B, Feeney J & Rob- erts GCK (1979) Stereochemistry of reduction of folic acid using dihydrofolate reductase. Chem Commun 15, 922–924. 36 Sawaya MR & Kraut J (1997) Loop and subdomain movements in the mechanism of Escherichia coli dihy- drofolate reductase: crystallographic evidence. Biochem- istry 36, 586–603. 37 Iwakura M, Jones BE, Luo JB & Matthews CR (1995) A strategy for testing the suitability of cysteine replace- ments in dihydrofolate reductase from Escherichia coli. J Biochem 117, 480–488. 38 Greene RF & Pace CN (1974) Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, a-chymotrypsin, and b-lactoglobulin. J Biol Chem 249, 5388–5393. 39 Loveridge EJ, Tey LH & Allemann RK (2010) Solvent effects on catalysis by Escherichia coli dihydrofolate reductase. J Am Chem Soc 132, 1137–1143. 40 Swanwick RS, Shrimpton PJ & Allemann RK (2004) Pivotal role of Gly 121 in dihydrofolate reductase from Escherichia coli: the altered structure of a mutant enzyme may form the basis of its diminished catalytic performance. Biochemistry 43, 4119–4127. 41 Sham YY, Ma BY, Tsai CJ & Nussinov R (2002) Ther- mal unfolding molecular dynamics simulation of Escher- ichia coli dihydrofolate reductase: thermal stability of protein domains and unfolding pathway. Proteins 46, 308–320. 42 Frieden C (1990) Refolding of Escherichia coli dihydrof- olate-reductase – sequential formation of substrate binding sites. Proc Natl Acad Sci USA 87, 4413–4416. 43 Kuwajima K, Garvey EP, Finn BE, Matthews CR & Sugai S (2002) Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy. Biochemistry 30, 7693– 7703. 44 Jennings PA, Finn BE, Jones BE & Matthews CR (2002) A reexamination of the folding mechanism of di- hydrofolate reductase from Escherichia coli: verification and refinement of a four-channel model. Biochemistry 32, 3783–3789. 45 Jones BE, Beechem JM & Matthews CR (2002) Local and global dynamics during the folding of Escherichia coli dihydrofolate reductase by time- resolved fluorescence spectroscopy. Biochemistry 34, 1867–1877. 46 Antikainen NM, Smiley RD, Benkovic SJ & Hammes GG (2005) Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase. Biochemistry 44, 16835–16843. 47 Rajagopalan PTR, Zhang ZQ, McCourt L, Dwyer M, Benkovic SJ & Hammes GG (2002) Interaction of dihy- drofolate reductase with methotrexate: ensemble and single-molecule kinetics. Proc Natl Acad Sci USA 99, 13481–13486. 48 Venkitakrishnan RP, Zaborowski E, McElheny D, Benkovic SJ, Dyson HJ & Wright PE (2004) Conforma- tional changes in the active site loops of dihydrofolate reductase during the catalytic cycle. Biochemistry 43, 16046–16055. 49 Gekko K, Kunori Y, Takeuchi H, Ichihara S & Kodama M (1994) Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important role of a flexible loop in the stability and function. J Biochem 116, 34–41. 50 Cameron CE & Benkovic SJ (1997) Evidence for a functional role of the dynamics of glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant. Biochemistry 36, 15792–15800. 51 Fierke CA, Johnson KA & Benkovic SJ (1987) Con- struction and evaluation of the kinetic scheme associ- ated with dihydrofolate reductase from Escherichia coli. Biochemistry 26, 4085–4092. Supporting information The following supplementary material is available: Fig. S1. MALDI-TOF MS following trypsin digestion of EcDHFR triple mutants. Fig. S2. CD spectra at 20 °C and thermal melting curves of WT EcDHFR and DM EcDHFR. Fig. S3. Urea denaturation curves and free energy of unfolding for WT EcDHFR and DM EcDHFR. Fig. S4. Binding curves for NADPH and folate with WT EcDHFR and DM EcDHFR. Fig. S5. CD spectra at 20 °C and thermal melting curves of EcDHFR triple mutants. Fig. S6. Free energy of unfolding at 20 °C for EcDHFR triple mutants. Glycosylation of E. coli DHFR L H. Tey et al. 2178 FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS Table S1. Mean residue ellipticities at 222 nm. Table S2. Midpoints of the urea-induced unfolding transition. Table S3. Gradients of the free energy of unfolding plots. Table S4. Dissociation constants for NADPH. Table S5. Dissociation constants for folate. Table S6. Hydride transfer rate constants. Table S7. Steady-state turnover rates. Table S8. Michaelis constants. Table S9. k cat ⁄ K M . This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. L H. Tey et al. Glycosylation of E. coli DHFR FEBS Journal 277 (2010) 2171–2179 ª 2010 The Authors Journal compilation ª 2010 FEBS 2179 . Highly site-selective stability increases by glycosylation of dihydrofolate reductase Lai-Hock Tey 1 , E. Joel Loveridge 1 ,. and stability is restored by glycosylation [14]. The change in stability is because of the glycan rather than the acetamide linkage [14]. Stability of the glycosylated