Effect of Betaine and Arginine on Interaction of αB-Crystallin with Glycogen Phosphorylase <i>b</i>

Protein–protein interactions (PPIs) play an important role in many biological processes in a living cell. Among them chaperone–client interactions are the most important. In this work PPIs of αB-crystallin and glycogen phosphorylase b (Phb) in the presence of betaine (Bet) and arginine (Arg) at 48 °C and ionic strength of 0.15 M were studied using methods of dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation. It was shown that Bet enhanced, while Arg reduced both the stability of αB-crystallin and its adsorption capacity (AC0) to the target protein at the stage of aggregate growth. Thus, the anti-aggregation activity of αB-crystallin increased in the presence of Bet and decreased under the influence of Arg, which resulted in inhibition or acceleration of Phb aggregation, respectively. Our data show that chemical chaperones can influence the tertiary and quaternary structure of both the target protein and the protein chaperone. The presence of the substrate protein also affects the quaternary structure of αB-crystallin, causing its disassembly. This is inextricably linked to the anti-aggregation activity of αB-crystallin, which in turn affects its PPI with the target protein. Thus, our studies contribute to understanding the mechanism of interaction between chaperones and proteins

Effect of Arginine on Chaperone-Like Activity of HspB6 and Monomeric 14-3-3ζ

The effect of protein chaperones HspB6 and the monomeric form of the protein 14-3-3&zeta; (14-3-3&zeta;m) on a test system based on thermal aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) at 37 &deg;C and a constant ionic strength (0.15 M) was studied using dynamic light scattering. A significant increase in the anti-aggregation activity of HspB6 and 14-3-3&zeta;m was demonstrated in the presence of 0.1 M arginine (Arg). To compare the effects of these chaperones on UV-Phb aggregation, the values of initial stoichiometry of the chaperone&ndash;target protein complex (S0) were used. The analysis of the S0 values shows that in the presence of Arg fewer chaperone subunits are needed to completely prevent aggregation of the UV-Phb subunit. The changes in the structures of HspB6 and 14-3-3&zeta;m induced by binding of Arg were evaluated by the fluorescence spectroscopy and differential scanning calorimetry. It was suggested that Arg caused conformational changes in chaperone molecules, which led to a decrease in the thermal stability of protein chaperones and their destabilization

Effect of Chemical Chaperones on the Stability of Proteins during Heat– or Freeze–Thaw Stress

The importance of studying the structural stability of proteins is determined by the structure–function relationship. Protein stability is influenced by many factors among which are freeze–thaw and thermal stresses. The effect of trehalose, betaine, sorbitol and 2-hydroxypropyl-β-cyclodextrin (HPCD) on the stability and aggregation of bovine liver glutamate dehydrogenase (GDH) upon heating at 50 °C or freeze–thawing was studied by dynamic light scattering, differential scanning calorimetry, analytical ultracentrifugation and circular dichroism spectroscopy. A freeze–thaw cycle resulted in the complete loss of the secondary and tertiary structure, and aggregation of GDH. All the cosolutes suppressed freeze–thaw- and heat-induced aggregation of GDH and increased the protein thermal stability. The effective concentrations of the cosolutes during freeze–thawing were lower than during heating. Sorbitol exhibited the highest anti-aggregation activity under freeze–thaw stress, whereas the most effective agents stabilizing the tertiary structure of GDH were HPCD and betaine. HPCD and trehalose were the most effective agents suppressing GDH thermal aggregation. All the chemical chaperones stabilized various soluble oligomeric forms of GDH against both types of stress. The data on GDH were compared with the effects of the same cosolutes on glycogen phosphorylase b during thermal and freeze–thaw-induced aggregation. This research can find further application in biotechnology and pharmaceutics

Change in the content of the main elements of the secondary structure of glycogen phosphorylase <i>b</i> after UV irradiation.

<p>Change in the content of the main elements of the secondary structure of glycogen phosphorylase <i>b</i> after UV irradiation.</p

The changes in the size of the protein aggregates formed in the process of UV-Ph<i>b</i> aggregation at 37°C.

<p>(A and B) The distribution of particles by their size registered for UV-Ph<i>b</i> (0.4 mg/ml) heated for 0.5 and 13.5 min, respectively. (C) The dependences of the hydrodynamic radius (<i>R</i>_h) on time for small-sized (1) and large-sized aggregates (2) ([UV-Ph<i>b</i>] = 0.5 mg/ml). (D) The <i>R</i>_h,2 value versus γ_agg plots obtained at the following concentrations of UV-Ph<i>b</i>: 0.15 (1), 0.3 (2), 0.5 (3) and 1.2 mg/ml (4). <i>R</i>_h,2 is the hydrodynamic radius of large-sized aggregates. γ_agg is the fraction of the aggregated protein calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e012" target="_blank">Eq 9</a> (0.03 M Hepes buffer, pH 6.8, containing 0.1 M NaCl; radiation dose was 9.4 J/cm²).</p

A thermal after-effect of UV irradiation of muscle glycogen phosphorylase <i>b</i>

<div><p>Different test systems are used to characterize the anti-aggregation efficiency of molecular chaperone proteins and of low-molecular-weight chemical chaperones. Test systems based on aggregation of UV-irradiated protein are of special interest because they allow studying the protective action of different agents at physiological temperatures. The kinetics of UV-irradiated glycogen phosphorylase <i>b</i> (UV-Ph<i>b</i>) from rabbit skeletal muscle was studied at 37°C using dynamic light scattering in a wide range of protein concentrations. It has been shown that the order of aggregation with respect to the protein is equal to unity. A conclusion has been made that the rate-limiting stage of the overall process of aggregation is heat-induced structural reorganization of a UV-Ph<i>b</i> molecule, which contains concealed damage.</p></div

Enzymatic activity and heat stability of UV-Ph<i>b</i>.

<p>(A) Dependence of the relative enzymatic activity <i>A</i>/<i>A</i>₀. (B) Dependences of the excess heat capacity () on temperature for intact Ph<i>b</i> and Ph<i>b</i> irradiated by different UV doses. The initial concentration of Ph<i>b</i> was 1.5 mg/ml. was calculated per Ph<i>b</i> dimer (<i>M</i>_r = 194800 Da). (C) Position of the maximum <i>T</i>_max on the DSC profiles. (D) Relative denaturation heat <i>Q</i>/<i>Q</i>₀. Conditions of the experiments: 0.03 M Hepes buffer, pH 6.8, containing 0.1 M NaCl. <i>A</i>₀ and <i>A</i> are the values of enzymatic activity for intact Ph<i>b</i> and UV-Ph<i>b</i>, respectively. <i>Q</i>₀ and <i>Q</i> are the values of the area under DSC profile for intact Ph<i>b</i> and UV-Ph<i>b</i>, respectively. Three independent measurements were used to determine the arrow bars shown in this figure.</p

Scheme illustrating thermal after-effect of UV irradiation of Ph<i>b</i>.

<p>Heating of UV-Ph<i>b</i> with concealed damage (P⁰) at 37°C results in a structural reorganization of UV-irradiated protein to a state with manifested damage (P^a). Slow transformation of P⁰ into P^a is followed by the nucleation stage and fast stage of aggregate growth with formation of amorphous aggregates. The growth of aggregates proceeds by attachment of P^a to the existing aggregates (basic aggregation pathway). Sticking of protein aggregates can be observed in the course of accumulation of large-sized aggregates (additional aggregation stage).</p

A comparison of the properties of intact Ph<i>b</i> and UV-Ph<i>b</i> using CD.

<p>(A) The CD spectra for intact Ph<i>b</i> (1 mg/ml, curve 1) and Ph<i>b</i> irradiated with the dose of 9.4 J/cm² (1 mg/ml, curve 2) at 10°C. (B) The dependence of ellipticity at 220 nm and (C) the portion of unfolded protein (γ_U) on temperature for intact Ph<i>b</i> (0.17 mg/ml, curve 1) and UV-Ph<i>b</i> (0.17 mg/ml, curve 2). The values of γ_U were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e011" target="_blank">Eq 8</a>. The dashed horizontal lines on panel B correspond to the asymptotes for the regions of relatively low and relatively high temperatures. The dashed horizontal lines on panel C correspond to the γ_U values equal to 0, 0.5 and 1.0.</p

Sedimentation behaviour of UV-Ph<i>b</i> (0.75 mg/ml) at 25°C.

<p>The samples were heated at 37°C for 90 min and cooled to 25°C after heating. (A) Sedimentation profiles for Ph<i>b</i> irradiated with dose of 9.4 (sample 1) and 12.5 J/cm² (sample 2) before heating. <i>A</i>₂₈₈ is absorbance at 288 nm, <i>r</i> is radial distance. Control curves (control 1 and control 2) correspond to non-heated UV-Ph<i>b</i> (radiation doses were 9.4 and 12.5 J/cm², respectively). Sedimentation runs were carried out at 25°C. Rotor speed was 15,000 rpm. (B) Sedimentation profiles for sample 1 (Ph<i>b</i> irradiated with dose of 9.4 J/cm² before heating) at 25°C. Rotor speed was 48,000 rpm. Every 4th scan was taken for presentation. (C) The <i>c</i>(<i>s</i>) sedimentation coefficient distributions obtained at 25°C were transformed to standard <i>s</i>_20,w distributions for samples 1 and 2. Sedimentation runs were carried out at 25°C. Rotor speed was 48,000 rpm.</p