Effect of arginine on stability and aggregation of muscle glycogen phosphorylase b.

Arginine (Arg) is frequently used in biotechnology and pharmaceutics to stabilize protein preparations. When using charged ions like Arg, it is necessary to take into account their contribution to the increase in ionic strength, in addition to the effect of Arg on particular processes occurring under the conditions of constancy of ionic strength. Here, we examined contribution of ionic strength (0.15 and 0.5 M) to the effects of Arg on denaturation, thermal inactivation and aggregation of skeletal muscle glycogen phosphorylase b (Phb). Dynamic light scattering, analytical ultracentrifugation, differential scanning calorimetry, circular dichroism and enzymatic activity assay were used to assess the effects of Arg at constant ionic strength compared with the effects of ionic strength alone. We found that high ionic strength did not affect the secondary structure of Phb, but changed conformation of the protein. Such a destabilization of the enzyme causes an increase in the initial rate of aggregation and inactivation of Phb thereby affecting its denaturation. Binding of Arg causes additional changes in the protein conformation, weakening the bonds between monomers in the dimer. This causes the dimer to dissociate into monomers, which rapidly aggregate. Thus, Arg acts on these processes much stronger than just ionic strength.

The role of vibronic modes in formation of red antenna states of cyanobacterial PSI.

Cyanobacterial photosystem I (PSI) constitutes monomeric and trimeric pigment-protein complexes whose optical properties are marked by the presence of long-wavelength absorption bands. In spite of numerous experimental studies, the nature of these bands is still under debate and requires intensive theoretical analysis. Collecting together the data of linear spectroscopy and single-molecule spectroscopy (SMS) of PSI from Arthrospira platensis, we performed quantum modeling of the optical response based on molecular exciton theory (ET) and the multimode Brownian oscillator model (MBOM). Applying MBOM, the spectra of the red antenna state were calculated considering a particular for each red state adjustment of the low-frequency vibronic modes. Within the framework of our PSI exciton model it was shown that the coupling energy between antenna chlorophylls cannot be a factor of the red states formation, thus the long-wavelength bands are calculated without attribution to so-called antenna red chlorophylls. By the fitting of Huang-Rhys factors and frequencies for the lowest vibronic modes, we were able to reproduce the effects of strong and weak electron-phonon coupling experimentally observed in SMS spectra of red antenna states. Based on our theoretical calculations and also analysis of existing crystal structures of cyanobacterial PSI, we assumed that long-wavelength Chls can be localized in the peripheral protein subunits containing one or two pigment molecules.

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 14-3-3ζ (14-3-3ζm) on a test system based on thermal aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) at 37 °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 14-3-3ζm 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-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 14-3-3ζm 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.

Comparative effects of trehalose and 2-hydroxypropyl-ß-cyclodextrin on aggregation of UV-irradiated muscle glycogen phosphorylase b.

Chemical chaperones are a class of small molecules which enhance folding and prevent aggregation of proteins. Investigation of their effects on the processes of protein aggregation is of importance for further understanding of implication of protein aggregation in neurodegenerative diseases, as well as for solving biotechnological tasks. The effects of chemical chaperones trehalose and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on the kinetics of aggregation of UV-irradiated muscle glycogen phosphorylase b (UV-Phb) at 37 °C have been studied. The process of thermal aggregation of UV-Phb includes a slow stage of structural reorganization of the UV-Phb molecule, nucleation stage and fast attachment of structurally reorganized UV-Phb molecules to nuclei formed during the nucleation stage. It was shown that both trehalose and HP-ß-CD increased the duration of the nucleation phase and slowed down the rate of structural reorganization of the UV-Phb molecule. This conclusion has been confirmed by the circular dichroism data. In the absence of chaperones, 82% UV-Phb aggregates, whereas in the presence of HP-ß-CD or trehalose the portion of aggregated protein decreases to 70 and 66%, respectively. The data on analytical ultracentrifugation demonstrated that in the presence of these additives the size of protein aggregates decreased. Analysis of the combined effect of trehalose and HP-ß-CD on UV-Phb aggregation showed that protein aggregation was independently affected by trehalose and HP-ß-CD.

Effect of ionic strength and arginine on aggregation of UV-irradiated muscle glycogen phosphorylase b.

In this work the effect of ionic strength and arginine on the kinetics of aggregation of UV-irradiated muscle glycogen phosphorylase b (UV-Phb) was studied using dynamic light scattering at 37 °C at various ionic strengths (0.02-0.7 M). Under these conditions the rate-limiting stage of the overall aggregation process is the structural reorganization of UV-Phb, which can be characterized by the first order rate constant kI. It was shown that an increase in NaCl concentration caused a decrease in the kI value, suggesting a slowdown of the UV-Phb structural reorganization. Circular dichroism data confirmed this conclusion. Arginine is widely used in biotechnology as an agent suppressing protein aggregation. However, arginine is a charged molecule, and, when studying the action of arginine on protein aggregation, the effects of ionic strength should be taken into account. To evaluate the effect of arginine, experiments were conducted at fixed values of ionic strength (0.15 M and 0.5 M). It was shown that at a low ionic strength arginine (0-0.13 M) accelerated the process of protein aggregation, whereas at higher ionic strength arginine (0-0.48 M) acted as an aggregation suppressor.

A thermal after-effect of UV irradiation of muscle glycogen phosphorylase b.

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 b (UV-Phb) 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-Phb molecule, which contains concealed damage.

Thermostability of photosystem I trimers and monomers from the cyanobacterium Thermosynechococcus elongatus.

The performance of solar energy conversion into alternative energy sources in artificial systems highly depends on the thermostability of photosystem I (PSI) complexes Terasaki et al. (2007), Iwuchukwu et al. (2010), Kothe et al. (2013) . To assess the thermostability of PSI complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus heating induced perturbations on the level of secondary structure of the proteins were studied. Changes were monitored by Fourier transform infrared (FT-IR) spectra in the mid-IR region upon slow heating (1°C per minute) of samples in D2O phosphate buffer (pD 7.4) from 20°C to 100°C. These spectra showed distinct changes in the Amide I region of PSI complexes as a function of the rising temperature. Absorbance at the Amide I maximum of PSI monomers (centered around 1653cm-1), gradually dropped in two temperature intervals, i.e. 60-75 and 80-90°C. In contrast, absorbance at the Amide I maximum of PSI trimers (around 1656cm-1) dropped only in one temperature interval 80-95°C. The thermal profile of the spectral shift of α-helices bands in the region 1656-1642cm-1 confirms the same two temperature intervals for PSI monomers and only one interval for trimers. Apparently, the observed absorbance changes at the Amide I maximum during heating of PSI monomers and trimers are caused by deformation and unfolding of α-helices. The absence of absorbance changes in the interval of 20-65°C in PSI trimers is probably caused by a greater stability of protein secondary structure as compared to that in monomers. Upon heating above 80°C a large part of α-helices both in trimers and monomers converts to unordered and aggregated structures. Spectral changes of PSI trimers and monomers heated up to 100°C are irreversible due to protein denaturation and non-specific aggregation of complexes leading to new absorption bands at 1618-1620cm-1. We propose that monomers shield the denaturation sensitive sides at the monomer/monomer interface within a trimer, making the oligomeric structure more stable against thermal stress.

Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin.

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.

Quantum yield of P700+ photodestruction in isolated photosystem I complexes of the cyanobacterium Arthrospira platensis.

The photostability of P700 cation radical (P700+) was studied by evaluating the quantum yields of P700(+) photodestruction in photosystem I (PSI) complexes of the cyanobacterium Arthrospira platensis. The time courses of P700+ photodestruction in PSI trimers and monomers have been measured in aerobic conditions under selective excitation of far-red absorption band of P700+ by intense light of laser diodes. Long-term exposure of PSI complexes to 808 or 870 nm laser light caused destruction of P700+ and antenna chlorophylls. The true integral quantum yield of P700+ photodestruction calculated from these data was less than 0.7-1.4 x 10(-8). Illumination of PSI complexes by 650 nm light caused destruction of antenna chlorophylls with true quantum yield of about 6-7 x 10(-6) and damage of P700 with apparent quantum yield 2-3 x 10(-8). Preferential photodestruction of the long-wavelength antenna chlorophyll absorbing at 710 nm as compared with bulk chlorophylls was observed. About three orders of difference in magnitude between quantum yields of P700+ and bulk chlorophyll photodestruction indicates that P700+ is extremely photostable for functioning as an efficient quencher of singlet excitation energy in PSI.

Steady-state and transient polarized absorption spectroscopy of photosystem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus.

Core antenna and reaction centre of photosystem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(-), 683-685(-), 696-697(-), and 711(-) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700(+)A(1)(-) or (3)P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P(A), whereas the cation is localized most likely on P(B).