Dissociation and unfolding of the pyruvate dehydrogenase complex by guanidinium chloride

West, Shauna; Rice, Jacqueline; Beaumont, Ellen S.; Kelly, Sharon M.; Price, Nicholas C.; Lindsay, J. Gordon

doi:10.1042/bj3081025

Cited by 6 publications

(4 citation statements)

References 31 publications

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“…The formation of an intermediate trimeric species in the analytical ultracentrifuge has been described [21, 22], but no corresponding peak was observed in the present experiments. Previous fluorimetric studies [23]of the icosahedral bovine heart PDH complex have also suggested that the 60‐mer and trimeric E2p species dissociate into monomers, with 50% loss of tertiary structure at 1.9 M GuHCl and 50% loss of secondary structure at 2.8 M GuHCl.…”

Section: Resultsmentioning

confidence: 89%

The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Allen

Perham

1997

FEBS Letters

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A sub-gene encoding the catalytic (acetyltransferase) domain (E2pCD) comprising residues 173-427 of the dihydrolipoyl acetyltransferase (E2p) chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was expressed in Escherichia coli. The product assembled to form the characteristic icosahedral (60-mer) core structure with full catalytic activity. The K m values for dihydrolipoamide and acetyl-CoA were 1.2 mM and 13 jiM, respectively. Dissociation of the icosahedral E2pCD into monomers by exposure to guanidine hydrochloride and the subsequent reassociation by gradual removal of the denaturing agent demonstrated the ability of the polypeptide chain to fold and reassemble in the absence of chaperonins.

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Section: Resultsmentioning

confidence: 89%

The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Allen

Perham

1997

FEBS Letters

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“…Research has shown that the PDC can be dissassembled in the presence of chaotropic agents such as guanidinium–HCl. 56 We have found that the addition of 1 M urea to CFPS reaction mixtures prior to loading onto the sucrose gradient results in little or no co-purification of the PDC proteins along with assembled VLPs (data not shown).…”

Section: Resultsmentioning

confidence: 90%

Surface Functionalization of Virus-Like Particles by Direct Conjugation Using Azide−Alkyne Click Chemistry

2011

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We present a cell-free protein synthesis (CFPS) platform and a one-step, direct conjugation scheme for producing virus-like particle (VLP) assemblies that display multiple ligands including proteins, nucleic acids, and other molecules. Using a global methionine replacement approach, we produced bacteriophage MS2 and bacteriophage Qβ VLPs with surface-exposed methionine analogues (azidohomoalanine and homopropargylglycine) containing azide and alkyne side chains. CFPS enabled the production of VLPs with yields of ~300 μg/mL and with 85% incorporation of methionine analogues without requiring a methionine auxotrophic production host. We then directly conjugated azide- and alkyne-containing proteins (including an antibody fragment and the granulocyte-macrophage colony stimulating factor, or GM-CSF), nucleic acids and poly(ethylene glycol) chains to the VLP surface using Cu(I) catalyzed click chemistry. The GM-CSF protein, after conjugation to VLPs, was shown to partially retain its ability to stimulate the proliferation of cells. Conjugation of GM-CSF to VLPs resulted in a 3–5-fold reduction in its bioactivity. The direct attachment scheme facilitated conjugation of three different ligands to the VLPs in a single step, and enabled control of the relative ratios and surface abundance of the attached species. This platform can be used for the production of novel VLP bioconjugates for use as drug delivery vehicles, diagnostics, and vaccines.

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“…It is generally recognized that most enzymes will lose their catalytic activity in denaturant solutions (Zhou et al 1993; West et al 1995), but the AK from shrimp exhibited a small increase in specific activity in low‐concentration urea (France and Grossman 1996). Most AKs are 40‐kDa monomers, and the other PKs—CK, GK (glucokinase), and LK—are dimeric, or octameric in the case of mitochondrial CK (Fritz‐Wolf et al 1996).…”

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confidence: 99%

Unassisted refolding of urea‐denatured arginine kinase from shrimp Feneropenaeus chinensis: Evidence for two equilibrium intermediates in the refolding pathway

Pan¹,

Yu²,

Su³

et al. 2004

Protein Science

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The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and NЈ) were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (NЈ) existed between a 0.4-and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K D ) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.

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Dissociation and unfolding of the pyruvate dehydrogenase complex by guanidinium chloride

Cited by 6 publications

References 31 publications

The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Surface Functionalization of Virus-Like Particles by Direct Conjugation Using Azide−Alkyne Click Chemistry

Unassisted refolding of urea‐denatured arginine kinase from shrimp Feneropenaeus chinensis: Evidence for two equilibrium intermediates in the refolding pathway

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