R.A. Asryants

Determination of sepharose-bound protein with coomassie brilliant blue G-250

The colorimetric procedure of Bradford (M.M. Bradford, 1976, Anal. Biochem. 72, 248-254) was found to be convenient for determining the content of a protein immobilized on Sepharose. Being simple, sensitive, and rapid, this method appears very useful in studies involving multiple analyses of immobilized protein species present at low concentrations.

Evidence for the formation of start aggregates as an initial stage of protein aggregation.

The kinetics of thermal aggregation of glycogen phosphorylase b and glyceraldehyde 3‐phosphate dehydrogenase from rabbit skeletal muscles were studied using dynamic light scattering. Use of high concentrations of the enzymes (1–3 mg/ml) provided a simultaneous registration of the native enzyme forms and protein aggregates. It was shown that initially registered aggregates (start aggregates) were large‐sized particles. The hydrodynamic radius of the start aggregates was about 100 nm. The intermediate states between the native enzyme forms and start aggregates were not detected. The initial increase in the light scattering intensity is connected with accumulation of the start aggregates, the size of the latter remaining unchanged. From a certain moment in time aggregates of higher order, formed as a result of sticking of the start aggregates, make a major contribution to the enhancement of the light scattering intensity.

Kinetics of aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle. Effect of agents possessing chaperone-like activity

An aggregation test system based on the aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle has been proposed. On the basis of the measurements of the enzyme activity and differential scanning calorimetry data a conclusion has been made that UV radiation results in formation of damaged protein molecules with lower thermostability. It was shown that the order of aggregation rate for UV-irradiated GAPDH with respect to the protein was close to 2. This means that such a test system allows detecting the effect of various agents exclusively on the stage of aggregation of unfolded protein molecules. The influence of α-crystallin and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) on aggregation of UV-irradiated GAPDH was studied. Despite the fact that HP-β-CD accelerates thermal aggregation of non-irradiated GAPDH, in the case of aggregation of UV-irradiated GAPDH HP-β-CD reveals a purely protective effect.

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH‐groups modified by 5,5′‐dithiobis(2‐nitrobenzoate); (2) O‐R‐type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O‐P‐type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O‐R‐type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants Kd of 0.4 and 0.9 μM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg‐ATP. Chemically modified GAPDH and the O‐R‐type mutant dimers inhibited GroEL‐assisted refolding of urea‐denatured wild‐type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O‐R‐dimers, the O‐P‐type mutant dimers neither bound nor affected GroEL‐assisted refolding of the wild‐type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.

Essential arginine residues in d-glyceraldehyde-3-phosphate dehydrogenase

Two arginyl residues per subunit of yeast D-glyceraldehyde-3-phoshphate dehydrogenase were modified by treatment with butanedione without significant changes in the compostion of other amino acid residues. The modified enzyme displays no dehydrogenase activity. It retains the capacity for interacting with the coenzyme NAD, but binds it less firmly than does the native enzyme. The molar absorbance of the enzyme-NAD complex is markedly reduced and the reactivity of the active-center SH groups is changed in the modified enzyme. The native and modified enzymes show identical fluorescence spectra, absorbance and CD spectra.

Thermal unfolding of phosphorylating d-glyceraldehyde-3-phosphate dehydrogenase studied by differential scanning calorimetry

Thermal unfolding parameters were determined for a two-domain tetrameric enzyme, phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for its isolated NAD(+)-binding domain. At pH 8.0, the transition temperatures (t(max)) for the apoforms of the native Bacillus stearothermophilus GAPDH and the isolated domain were 78.3 degrees C and 61.9 degrees C, with calorimetric enthalpies (DeltaH(cal)) of 4415 and 437 kJ/mol (or 30.7 and 22.1 J/g), respectively. In the presence of nearly saturating NAD(+) concentrations, the t(max) and the DeltaH(cal) increased by 13.6 degrees C and by 2365 kJ/mol, respectively, for the native apoenzyme, and by 2.8 degrees C and 109 kJ/mol for the isolated domain. These results indicate that interdomain interactions are essential for NAD(+) to produce its stabilizing effect on the structure of the native enzyme. The thermal stability of the isolated NAD(+)-binding domain increased considerably upon transition from pH 6.0 to 8.0. By contrast, native GAPDH exhibited greater stability at pH 6.0; similar pH-dependencies of thermal stability were displayed by GAPDHs isolated from rabbit muscle and Escherichia coli. The binding of NAD(+) to rabbit muscle apoenzyme increased t(max) and DeltaH(cal) and diminished the widths of the DSC curves; the effect was found to grow progressively with increasing coenzyme concentrations. Alkylation of the essential Cys149 with iodoacetamide destabilized the apoenzyme and altered the effect of NAD(+). Replacement of Cys149 by Ser or by Ala in the B. stearothermophilus GAPDH produced some stabilization, the effect of added NAD(+) being basically similar to that observed with the wild-type enzyme. These data indicate that neither the ion pairing between Cys149 and His176 nor the charge transfer interaction between Cys149 and NAD(+) make any significant contribution to the stabilization of the enzymes native tertiary structure and the accomplishment of NAD(+)-induced conformational changes. The H176N mutant exhibited dramatically lower heat stability, as reflected in the values of both DeltaH(cal) and t(max). Interestingly, NAD(+) binding resulted in much wider heat capacity curves, suggesting diminished cooperativity of the unfolding transition.

Effect of 2-hydroxypropyl-β-cyclodextrin on thermal inactivation, denaturation and aggregation of glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle.

The effect of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on thermal aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle at 45 degrees C has been studied using dynamic light scattering. In the presence of HP-beta-CD higher values of the rate of aggregation and larger aggregates were registered. The acceleration of GAPDH aggregation was due to destabilization of the enzyme molecule under the action of HP-beta-CD. This is evidenced by the data on thermal inactivation of GAPDH and differential scanning calorimetry.

Rabbit muscle tetrameric d-glyceraldehyde-3-phosphate dehydrogenase is locked in the asymmetric state by chemical modification of a single arginine per subunit

Modification of a single arginine residue per subunit of rabbit muscle tetrameric D-glyceraldehyde-3-phosphate dehydrogenase by 2,3-butanedione converts the enzyme into the form which retains 5-7% of the original activity and manifests cooperative properties that are absent in the native enzyme. It exhibits half-of-the-sites reactivity towards the natural substrate D-glyceraldehyde-3-phosphate. Titration of the modified enzyme with DTNB reveals only two instantaneously reacting SH groups, the total amount of SH groups approaching nine per tetramer. In the presence of 8 M urea, an additional seven SH groups become accessible to DTNB. This suggests that the arginine modification imposes some conformational constraints which affect the microenvironment of the active site cysteine residues in two subunits of the tetramer. The changes do not influence the interaction between the essential cysteine residue and NAD+ which is responsible for the change transfer complex formation, since the molar extinction coefficient of the apoenzyme-NAD+ complex, epsilon 360, was not altered upon the arginine modification. The native and close to four in the case of the native enzyme and about three with the modified one. The apparent pK values of Cys-149 within the functioning active centers of the tetramer were determined from the pH profiles of the inactivation rates in presence of iodoacetamide. The apparent pKa of the essential thiols was found to change upon enzyme modification from 9.44 to 10.07 in the apoenzyme and from 9.17 to 9.36 in the holoenzyme. The apparent pKa of the arginine residue determined from the pH dependence of the inactivation rate was equal to 9.0 and did not change upon apo-holo enzyme transition.

A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = K agg(T−T 0)2, where K agg is the parameter characterizing the initial rate of aggregation and T 0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T 0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets.

Role of thermoinduced dissociation in interaction between α-crystallin as an oligomeric chaperone and glyceraldehyde-3-phosphate dehydrogenase as an oligomeric protein substrate

The key function of small heat shock proteins(sHsp), a class of molecular chaperones, is the prevention of aggregation of denatured proteins. The chaperonlike activity of sHsp is determined by their interaction with unfolded protein substrates formed understress conditions (e.g., in stress induced by heat orultraviolet radiation).The proteins of the sHsp family are oligomers consisting of subunits with a molecular weight of 12–42kDa. The molecular weight of sHsp oligomers usuallyvaried in the range from 150 to 800 kDa. The keystructural characteristic of sHsp oligomers is theirability to undergo reversible dissociation at elevatedtemperatures. This yields oligomeric forms of asmaller size, in which the recognition sites for proteinsubstrates open [1–3]. Thus, dissociation of sHsp oligomers may be a necessary conditions for the expression of chaperonlike activity by small heat shock proteins [1–4]. This assumption was confirmed usingHsp26 from