Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

Abstract

Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.Keywords: dimerization; folding; oligomerization; thymidylate synthase Thymidylate synthase (TS) is a dimer of two identical, 35-kDa subunits (Kinemage 1) that catalyzes the conversion of deoxyuridine monophosphate (dUMP) and 5,lO-methylenetetrahydrofolate (CH2H4folate) to dTMP and 7,8-dihydrofolate (H2folate). TS provides the only de novo pathway to deoxythymidine monophosphate (dTMP) production and has been studied extensively from the standpoints of structure, function, and inhibition (Santi & Danenberg, 1984). The crystal structure, which has been determined to 2.3 A resolution for Lactobacillus casei TS (Hardy et al., 1987;Perry et al., 1990; Finer-Moore, unpubl.), shows that the monomers form dimer contacts primarily between two, five-stranded beta sheets that maintain a unique +28" dihedral angle between strand directions (Kinemage 4). Three of these strands form abeta kink that creates part of the active site pocket of the second monomer. Further, two active site arginine residues (178' and 179') are donated from the opposing monomer and coordinate with the phosphate moiety of the substrate (Kinemage 3). Thus, the dimeric structure of TS is essential for the function of the enzyme.In order to study interactions at the dimer interface of TS, we sought conditions that would allow the separation, unfolding, and reassembly of subunits. To date, there is no evidence that the dimer can be dissociated and reformed. In the present work, we show that TS can be denatured to unfolded monomers in urea and subsequently refolded to its catalytically active native state. Results and discussionThe concentration of urea required to unfold TS was determined using UV and CD spectroscopies. The difference in environment of tryptophan residues between native and unfolded protein was monitored by the UV absorbance change at 294 nm (Fig. 1). Three zones were apparent along the equilibrium unfolding transition: (1) a range below 3.5 M urea where spectral changes were not observed, (2) a sharp transition between 3.5 M and 5.5 M denaturant, and (3) a range above 5.5 M urea where no further change in absorbance was detected. The AGHZ0 calculated for this equilibrium unfolding transition at 0 M urea was 19.1 kcal mol" (see Materials and methods). To ascertain whether secondary structure was lost at high urea concentration, the CD spectrum of TS equilibrated in 8.0 M urea was compared with that of native protein (Fig. 2). With...

Complete Restoration of Activity to Inactive Mutants of Escherichia coli Thymidylate Synthase: Evidence that E. coli Thymidylate Synthase is a Half-the-Sites Activity Enzyme

“…Assuming that subunit exchange is involved in the restoration of TS activity, there would be only one cysteine containing active site in the resulting dimer, suggesting that only one complete active site is necessary to achieve full TS activity. When the extent of reactivation was measured by the urea denaturation-renaturation procedure of Perry et al (1992) that was used to restore activity to comparable mutant mixtures of L. casei TS , only half the E. coli wild-type activity was obtained, which is similar to what these investigators found.…”

Native Dimer Stabilizes the Subunit Tertiary Structure of Porcine Class pi Glutathione S-transferase

“…The sensitivity of enzyme activity to subtle structural changes in a protein molecule makes measuring this property a useful and widely used functional probe to monitor protein unfolding (Herold and Kirschner, 1990;Perry et al, 1992;Kwon et al, 1993;Zhuang et al, 1994). The sensitivity of enzyme activity to subtle structural changes in a protein molecule makes measuring this property a useful and widely used functional probe to monitor protein unfolding (Herold and Kirschner, 1990;Perry et al, 1992;Kwon et al, 1993;Zhuang et al, 1994).…”

Catalytically active cross-species heterodimers of thymidylate synthase

“…Up to 100 p L of each refolding reaction was assayed for TS activity in a total reaction volume of 1 mL. These conditions of unfolding/refolding and assay were optimized for L. casei TS (Perry et al, 1992).…”