Analysis of the reconstitution of oligomeric enzymes by crosslinking with glutaraldehyde: kinetics of reassociation of lactic dehydrogenase

Hermann, Reinhard; Jaenicke, Rainer; Rudolph, Rainer

doi:10.1021/bi00521a015

Cited by 91 publications

(73 citation statements)

References 35 publications

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“…However, 15% of the activity was regained upon a 10-fold, rapid dilution of the denatured protein into buffer. Other workers have observed that additives such as substrates, glycerol (Lee & Timasheff, 1977), or KC1 (Hermann et al, 1981) may enhance refolding of proteins into their native forms. Thus, refolding of urea-denatured TS was performed by rapid dilution in the presence of the putative a Unfolding-refolding experiments were performed in 20 mM KHP04, pH 7.0, 0.1 mM EDTA, 1 mM DTT at pH 7.0 and 4 "C plus the indicated additive.…”

Section: Resultsmentioning

confidence: 99%

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

et al. 1992

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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...

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Section: Resultsmentioning

confidence: 99%

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

et al. 1992

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“…This raises the question in such cases, which are not universal (cf. Hermann et al, 1981Hermann et al, , 1983, as to the nature of the conformation at the point of association. In the case of monomeric proteins, the intermediate that has been shown in many cases to precede the slow rate-limiting step to the native state has been termed the molten globule (Ptitsyn, 1992;Christensen and Pain, 1994).…”

Section: Discussionmentioning

confidence: 99%

The Folding and Assembly Pathway of Tumour Necrosis Factor TNFα, a Globular Trimeric Protein

Hlodan

Pain

1995

European Journal of Biochemistry

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The mechanisms of folding and assembly of the globular, trimeric protein tumour necrosis factor-a (TNF) were studied by chemical cross-linking. This revealed the rapid accumulation of a dimeric interrnediate. Under the conditions of renaturation used, formation of the trimer is complete within six minutes. The kinetics of change of intrinsic and 8-anilino-I-naphthalene sulfonic acid fluorescence are first order and, combined with the kinetics of association, reveal the presence of folding steps both before and subsequent to formation of the trimer. Results from gel exclusion chromatography and kinetics, together with the existence of an acid-induced molten globule, support the conclusion that TNF folds and assembles through a trimeric molten globule.

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“…The ratio of aggregation and tetramer formation was found to be kinetically controlled, with increasing aggregation at high enzyme concentrations or in the presence of moderate denaturant concentrations [5, 91. However, this mechanism does not explain the fact that the yield of reactivation is inversely related to the time of incubation at acid pH [12,16,171. Since acid dissociation is fast (as shown by light scattering or hybridization experiments [20]), the slow decrease in the yield of reactivation must be correlated with changes within the aciddissociated monomers. Hydrolysis of labile peptide bonds as a possible explanation is clearly excluded by dodecyl sulfate/polyacrylamide gel electrophoresis.…”

Section: Discussionmentioning

confidence: 99%

“…high concentrations of glycine or phosphate) slow down the transformation of the rapidly reactivating 'high-yield conformation' (M,) to the slowly reactivating 'low-yield conformation' (M,). Reversible precipitation of M, by 1 M Na,SO, can block the isomerization completely, so that maximum yields of reactivation can be obtained, even after several hours of incubation at acid pH [20].…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of Lactic Dehydrogenase after Acid Dissociation

Zettlmeissl

Rudolph

Jaenicke

1981

European Journal of Biochemistry

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Reconstitution of lactic dehydrogenase from pig muscle after dissociation at acidic pH produces native tetramers and inactive aggregates, both of which have been previously described with respect to their structural properties and the kinetics of their formation. The ratio of the two fractions is determined by the relative rates of reassociation and aggregation [G. ZettlmeiBI, R. Rudolph and R. Jaenicke (1979) Biochemistry 18, 5567-55711. The present study is concerned with the influence of residual structural elements within the acid-denatured monomers on the process of reconstitution.Long incubation at low pH leads to a marked decrease of the rate and yield of reactivation, caused by conformational changes within the partially unfolded monomers. These slow rearrangements are reflected by slight changes of the far-ultraviolet circular dichroism of the acid-dissociated monomers, indicating a decrease in helix content from 30 observed immediately after the transfer to low pH, to 25 % after completion of the acid transition.The unchanged near-ultraviolet circular dichroism and fluorescence properties prove local structural alterations to be of lesser importance compared to the changes of the residual backbone folding. Varying the experimental conditions during denaturation proves the slow decrease in the rate and yield of reactivation, as well as the changes in the far-ultraviolet circular dichroism to be enhanced with incubation time and temperature (0 -30

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Analysis of the reconstitution of oligomeric enzymes by crosslinking with glutaraldehyde: kinetics of reassociation of lactic dehydrogenase

Cited by 91 publications

References 35 publications

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

The Folding and Assembly Pathway of Tumour Necrosis Factor TNFα, a Globular Trimeric Protein

Reconstitution of Lactic Dehydrogenase after Acid Dissociation

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