A new mechanism of acyl group transfer in the reaction catalyzed by D-glyceraldehyde-3-phosphate dehydrogenase

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) catalyzes the oxidative phosphorylation of its substrate in a two-step reaction. Asa result of the first, oxidativestep, the covalent intermediate where in 3-phosphoglyceroyl moiety is bound to Cys149 of the active center is subjected to nucleophilic attack by inorganic phosphate, but remains resistant to hydrolytic decomposition. This ensures tight coupling of oxidation with phosphorylation in glycolysis. In this article, we present the experimental evidence for the conversion of GAPDH into a form capable of performing the reaction in the absence of inorganic phosphate. The structural basis for this conversion is the oxidation of a cysteine residue (probably Cys 153) into a sulfenic acid derivative under mild conditions to affect the integrity of the essential Cys 149. As a result, an intram olecular transfer of 3-phosphoglyceroyl group from the active center Cys 149 to Cys 153 becomes possible with subsequent hydrolysis of the sulfenyl carboxylate intermediate.     

Unusual spiral structures formed by glycated β-casein in the presence of thioflavin T: amyloid transformation?

1. Download : Download high-res image (188KB)
2. Download : Download full-size image

     

Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein–Protein Interactions

In the present work, we proposed to create special sorbents for the study of protein–protein interactions, based on the fixation of cysteine-inserted beta-casein mutants with thiol-Sepharose resin. As a model system, we used bovine beta-casein, which belongs to the family of intrinsically unstructured proteins. Insertion of distal cysteines into the unfolded protein was not found to significantly change beta-casein properties. An amphiphilic beta-casein molecule has one hydrophilic domain and one hydrophobic domain placed on the N- and C-terminus, thus enabling one to exploit its capacity to engage in different types of intermolecular interactions. Two different casein-Sepharose sorbents incorporating either C-4 or C-208 beta-casein mutants bound to thiol-Sepharose were produced, exposing the hydrophobic domain in the case of the C-4 and the hydrophilic domain in the case of the C-208 mutant, respectively. The results obtained using the proposed sorbents with native beta-casein, another partially unfolded protein prion, and an oligomeric globular glyceraldehyde-3-phosphate dehydrogenase were found to be consistent with the data obtained by ELISA on free protein–protein complexes. Thus, Sepharose modified with various proteins is suitable for isolation of proteins interacting with the chromatographic phase bound partners from multicomponent systems such as milk. The obtained results allow the proposing of a fast and convenient method to be used for isolation of proteins, determination of protein-interacting partners, and the study of multi-protein complexes.     

Interaction of polyanions with basic proteins, 2(a) : influence of complexing polyanions on the thermo-aggregation of oligomeric enzymes

The ability of synthetic polyanions to suppress thermo-aggregation of the oligomeric enzymes (glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and aspartate aminotransferase) has been established. The ability of the polyanions to reduce the thermo-aggregation increased in the order poly(methacrylic acid) < poly(acrylic acid) < sodium poly(styrene sulphonate), which agreed well with the increase, in the same order, of the charge density of the chains. The lengthening of the chains, as well as the rise in their relative content, resulted in an increase of the ability to reduce thermo-aggregation, mentioned above. Complete prevention of the enzyme aggregation was achieved when highly charged polyanions of a relatively high degree of polymerization were used in a concentration sufficient to solubilize the protein. Complexing with the polyanions prevented thermo-aggregation of the enzymes, but not their thermo-denaturation. The adverse effect of the complexing polyanions on the catalytic activity was reduced by the addition of a synthetic polycation, which resulted in a significant reactivation (up to 40%) of the enzyme. The possibility of preventing the thermo-aggregation of enzyme molecules and then partly restoring the enzyme activity, appears to be of particular interest when studying the aggregation mechanism of proteins that are prone to form the amyloid structures responsible for the development of neurodegenerative diseases like Alzheimer's disease, bovine spongiform encephalopathy and Huntington disease. This finding can also be considered as an important step in the creation of artificial chaperones.     

Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein–Protein Interactions

In the present work, we proposed to create special sorbents for the study of protein–protein interactions, based on the fixation of cysteine-inserted beta-casein mutants with thiol-Sepharose resin. As a model system, we used bovine beta-casein, which belongs to the family of intrinsically unstructured proteins. Insertion of distal cysteines into the unfolded protein was not found to significantly change beta-casein properties. An amphiphilic beta-casein molecule has one hydrophilic domain and one hydrophobic domain placed on the N- and C-terminus, thus enabling one to exploit its capacity to engage in different types of intermolecular interactions. Two different casein-Sepharose sorbents incorporating either C-4 or C-208 beta-casein mutants bound to thiol-Sepharose were produced, exposing the hydrophobic domain in the case of the C-4 and the hydrophilic domain in the case of the C-208 mutant, respectively. The results obtained using the proposed sorbents with native beta-casein, another partially unfolded protein prion, and an oligomeric globular glyceraldehyde-3-phosphate dehydrogenase were found to be consistent with the data obtained by ELISA on free protein–protein complexes. Thus, Sepharose modified with various proteins is suitable for isolation of proteins interacting with the chromatographic phase bound partners from multicomponent systems such as milk. The obtained results allow the proposing of a fast and convenient method to be used for isolation of proteins, determination of protein-interacting partners, and the study of multi-protein complexes.

     

Erratum to:Study of GroEL Conformational Mobility by Cryo-Electron Microscopy and Molecular Dynamics

Crystallography Reports - An Erratum to this paper has been published: https://doi.org/10.1134/S1063774521340058     

Light Scattering Study of the Antibody–Poly(methacrylic acid) and Antibody–Poly(acrylic acid) Conjugates in Aqueous Solutions

The effect of the conformational state of the polymer coil on the properties of protein–polymer conjugates has been studied for the conjugates of antibody (monoclonal antibody from 6C5 clone against inactivated rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase; Ab) with poly(methacrylic acid) (PMAA) or poly‐(acrylic acid) (PAA). The pH‐dependencies of molecular properties and structural parameters of aqueous solutions (radius of gyration, intensity of scattered light, hydrodynamic diameter, and polydispersity index) of Ab, PMAA, and PAA and their conjugates, i. e., Ab‐PMAA and Ab‐PAA, have been studied using static and dynamic light scattering techniques. While free Ab aggregates in solution and precipitates at its isoelectric point, the covalent attachment of a charged polymer to Ab prevents its association and shifts the precipitation point towards more acidic values (from pH 5.95 for Ab to pH ˜ 4.8 for Ab‐PMAA). The predominant role of the conformational status of the polymer in the process of conjugate precipitation has been considered. Contrary to the precipitation of Ab‐PMAA, the formation of stable colloidal particles was suggested for Ab‐PAA at pH < 4.8. In the conjugates, polymer chains surround the protein globule in an extremely compact manner while Ab significantly affects the polymer conformation. The essentially larger hydrodynamic radii of conjugates, when compared with their radii of gyration, confirm the strong interaction of conjugates with solvent molecules.     

Interaction of polyelectrolytes with proteins, 3. Influence of complexing polycations on the thermoaggregation of oligomeric enzymes

The ability of quaternized polyamines (poly-N-alkyl-4-vinylpyridinium bromides possessing a number, m, of methylene groups in the N-alkyl substituent or a degree of alkylation, beta, and n,n-ionene bromides) to suppress the thermoaggregation of glyceraldehyde-3-phosphate dehydrogenase increased in the order m = 1 < 3 < 5, beta = 95 < 85 < 70 < 45 < 35 < 20 and n = 3 < 6 < 10, which agrees well with the increase, in the same order, in the hydrophobicity of the chains. Complexing suppressed thermoaggregation, but not thermodenaturation of the enzyme, which was even encouraged by the polycations and occurred at room temperature when the most efficient suppressor (with beta = 20) was used. The adverse effect was reduced by the addition of sodium chloride which destroyed the complex and resulted in a noticeable reactivation.     

Methylglyoxal modification hinders amyloid conversion of prion protein

1. Download high-res image (138KB)
2. Download full-size image

     

D-glyceraldehyde-3-phosphate dehydrogenase

Examination of the properties ofEscherichia coli and rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenases (GPDHs) modified by 2,3-butanedione has shown that both tetrameric enzymes are stabilized, on selective modification of arginine residues (probably Arg 231), in an asymmetric state with only two active centers capable of performing the dehydrogenase reaction. The functionally incompetent active centers can be alkylated by iodoacetate or iodoacetamide in the case ofE. coli enzyme, but are inaccessible for these reagents in the case of rabbit muscle D-GPDH. These results are consistent with the idea that the two homologous enzymes share common principles of the protein design, but differ somewhat in their active centers geometries. Modification of the arginine produces marked changes in the shape of the charge transfer complex spectrum in the region of 300–370 nm, suggestive of the alterations in the microenvironment of the nicotinamide ring of NAD⁺, although the coenzyme binding characteristics remain largely unaltered. On arginine modification, the enzyme becomes insensitive to the effect of AMP on the kinetic parameters of p-nitrophenyl acetate hydrolysis reaction.