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

The comparison of the kinetics of aggregation of UV-Ph<i>b</i> (0.26 mg/ml) at 37°C followed by an increase in the concentration of aggregated UV-Ph<i>b</i> and increase in the light scattering intensity.

<p>(A) The time dependences of the concentration of aggregated UV-Ph<i>b</i> irradiated with the following doses: 7.5 (1), 9.4 (2) and 12.5 J/cm² (3). The points are the experimental data. The solid curve was calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e009" target="_blank">Eq 6</a> at the following values of parameters: <i>t</i>₀ = 6 min, <i>k</i>_I = 0.074 min^-1 and [UV-Ph<i>b</i>_agg]_lim = 0.16 mg/ml for curve 1; <i>t</i>₀ = 1.71 min, <i>k</i>_I = 0.107 min^-1 and [UV-Ph<i>b</i>_agg]_lim = 0.19 mg/ml for curve 2; <i>t</i>₀ = 0.67 min, <i>k</i>_I = 0.139 min^-1 and [UV-Ph<i>b</i>_agg]_lim = 0.21 mg/ml for curve 3. (B) The relationship between increment of the light scattering intensity (<i>I</i>–<i>I</i>₀) and the concentration of aggregated UV-Ph<i>b</i>. The radiation doses were the following: 7.5 (1), 9.4 (2) and 12.5 J/cm² (3). The concentration of aggregated UV-Ph<i>b</i> was determined from measurements of optical density of supernatant at 280 nm after precipitation of protein aggregates by centrifugation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#sec002" target="_blank">Methods</a>). The error bars were calculated using three independent measurements.</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

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

The kinetics of aggregation of UV-Ph<i>b</i> followed by the increase in the light scattering intensity at 37°C (radiation dose was 9.4 J/cm²).

<p>(A) The dependences of the light scattering intensity (<i>I–I</i>₀) on time obtained at the following concentrations of UV-Ph<i>b</i>: 0.2 (1), 0.4 (2), 0.6 (3), 0.9 (4) 1.2 (5) and 1.5 mg/ml (6). <i>I</i> and <i>I</i>₀ are the current and initial values of the light scattering intensity, respectively. (B) Fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e005" target="_blank">Eq 4</a> to the experimental data obtained at [UV-Ph<i>b</i>] = 0.4 mg/ml. Points are the experimental data. The solid curve was calculated at <i>k</i>_I = 0.115 min^-1 and <i>t</i>₀ = 5.54 min. The horizontal dashes correspond to <i>I</i>₀ and <i>I</i>_lim values. (C, D and E) The dependences of <i>k</i>_I, (<i>I</i>_lim−<i>I</i>₀) and <i>k</i>_I(<i>I</i>_lim−<i>I</i>₀) values on the concentration of UV-Ph<i>b</i>. (F) The kinetic curves represented in coordinates {[d(<i>I—I</i>₀)/d<i>t</i>]/[P]₀; (<i>I—I</i>₀)/[P]₀}, where [P]₀ is the concentration of UV-Ph<i>b</i>. The dimension of [d(<i>I—I</i>₀)/d<i>t</i>]/[P]₀ is [min^-1·(counts/s)]/(mg/ml); the dimension of (<i>I—I</i>₀)/[P]₀ is (counts/s)/(mg/ml). The concentrations of UV-Phb were the following: 0.2 (1), 0.4 (2), 0.6 (3), 0.9 mg/ml (4). The solid line was calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e006" target="_blank">Eq 5</a> at <i>k</i>_I = 0.122 min^-1 and ε = 1.67·10⁶ (counts/s)/(mg/ml). Three independent measurements were used to determine the error bars shown in panels C, D and E.</p

Structural plasticity and thermal stability of the histone-like protein from <i>Spiroplasma melliferum</i> are due to phenylalanine insertions into the conservative scaffold

<p>The histone-like (HU) protein is one of the major nucleoid-associated proteins of the bacterial nucleoid, which shares high sequence and structural similarity with IHF but differs from the latter in DNA-specificity. Here, we perform an analysis of structural-dynamic properties of HU protein from <i>Spiroplasma melliferum</i> and compare its behavior in solution to that of another mycoplasmal HU from <i>Mycoplasma gallisepticum</i>. The high-resolution heteronuclear NMR spectroscopy was coupled with molecular-dynamics study and comparative analysis of thermal denaturation of both mycoplasmal HU proteins. We suggest that stacking interactions in two aromatic clusters in the HUSpm dimeric interface determine not only high thermal stability of the protein, but also its structural plasticity experimentally observed as slow conformational exchange. One of these two centers of stacking interactions is highly conserved among the known HU and IHF proteins. Second aromatic core described recently in IHFs and IHF-like proteins is considered as a discriminating feature of IHFs. We performed an electromobility shift assay to confirm high affinities of HUSpm to both normal and distorted dsDNA, which are the characteristics of HU protein. MD simulations of HUSpm with alanine mutations of the residues forming the non-conserved aromatic cluster demonstrate its role in dimer stabilization, as both partial and complete distortion of the cluster enhances local flexibility of HUSpm.</p

Dependences of the light scattering intensity on time for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The dotted horizontal lines on the panels A, B and C correspond to <i>I</i>₂ values calculated from the dependences of the light scattering intensity on the portion of aggregated BSA.</p