Change in the Kinetic Regime of Aggregation of Yeast Alcohol Dehydrogenase in the Presence of 2-Hydroxypropyl-ß-cyclodextrin.

Chemical chaperones are low-molecular-weight compounds that suppress protein aggregation. They can influence different stages of the aggregation process-the stage of protein denaturation, the nucleation stage and the stage of aggregate growth-and this may lead to a change in the aggregation kinetic regime. Here, the possibility of changing the kinetic regime in the presence of a chemical chaperone 2-hydroxypropyl-ß-cyclodextrin (2-HP-ß-CD) was investigated for a test system based on the thermally induced aggregation of yeast alcohol dehydrogenase (yADH) at 56 °C. According to differential scanning calorimetry data, 2-HP-ß-CD did not affect the stage of the protein molecule unfolding. Dynamic light scattering data indicated changes in the aggregation kinetics of yADH during the nucleation and aggregate growth stages in the presence of the chaperone. The analysis of kinetic curves showed that the order of aggregation with respect to protein (nc), calculated for the stage of aggregate growth, changed from nc = 1 to nc = 2 with the addition of 100 mM 2-HP-ß-CD. The mechanism of 2-HP-ß-CD action on the yADH thermal aggregation leading to a change in its kinetic regime of aggregation is discussed.

The Effect of Chemical Chaperones on Proteins with Different Aggregation Kinetics.

Formation and accumulation of protein aggregates adversely affect intracellular processes in living cells and are negative factors in the production and storage of protein preparations. Chemical chaperones can prevent protein aggregation, but this effect is not universal and depends on the target protein structure and kinetics of its aggregation. We studied the effect of betaine (Bet) and lysine (Lys) on thermal aggregation of muscle glycogen phosphorylase b (Phb) at 48°C (aggregation order, n = 0.5), UV-irradiated Phb (UV-Phb) at 37°C (n = 1), and apo-form of Phb (apo-Phb) at 37°C (n = 2). Using dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation, we have shown that Bet protected Phb and apo-Phb from aggregation, but accelerated the aggregation of UV-Phb. At the same time, Lys prevented UV-Phb and apo-Phb aggregation, but increased the rate of Phb aggregation. The mechanisms of chemical chaperone action on the tertiary and quaternary structures and kinetics of thermal aggregation of the target proteins are discussed. Comparison of the effects of chemical chaperones on the proteins with different aggregation kinetics provides more complete information on the mechanism of their action.

Relationship between the Structure and Chaperone Activity of Human αA-Crystallin after Its Modification with Diabetes-Associated Oxidative Agents and Protective Role of Antioxidant Compounds.

The study was aimed to evaluate the impact of peroxynitrite (PON, oxidative stress agent in diabetes), methylglyoxal (MGO, diabetes-associated reactive carbonyl compound), and their simultaneous application on the structural and functional features of human αA-crystallin (αA-Cry) using various spectroscopy techniques. Additionally, the surface tension and oligomer size distribution of the treated and untreated protein were tested using tensiometric analysis and dynamic light scattering, respectively. Our results indicated that the reaction of PON and MGO with human αA-Cry leads to the formation of new chromophores, alterations in the secondary to quaternary protein structure, reduction in the size of protein oligomers, and significant enhancement in the chaperone activity of αA-Cry. To reverse the effects of the tested compounds, ascorbic acid and glutathione (main components of lens antioxidant defense system) were applied. As expected, the two antioxidant compounds significantly prevented formation of high molecular weight aggregates of αA-Cry (according to SDS-PAGE). Our results suggest that the lens antioxidant defense system, in particular, glutathione, may provide a strong protection against rapid incidence and progression of diabetic cataract by preventing the destructive reactions of highly reactive DM-associated metabolites.

Effect of Trehalose on Oligomeric State and Anti-Aggregation Activity of αB-Crystallin.

αB-Crystallin (αB-Cr), one of the main crystalline lens proteins, along with other crystallins maintains lens transparency suppressing protein aggregation and thus preventing cataractogenesis. αB-Cr belongs to the class of molecular chaperones; being expressed in many tissues it has a dynamic quaternary structure, which is essential for its chaperone-like activity. Shift in the equilibrium between ensembles of oligomers of different size allows regulating the chaperone activity. Trehalose is known to inhibit protein aggregation in vivo and in vitro, and it is widely used in biotechnology. The results of studying the effect of trehalose on the chaperone-like activity of crystallins can serve as a basis for the design of drugs delaying cataractogenesis. We have studied the trehalose effect on the quaternary structure and anti-aggregation activity of αB-Cr using muscle glycogen phosphorylase b (Phb) as a target protein. According to the dynamic light scattering data, trehalose affects the nucleation stage of Phb thermal aggregation at 48°C, and an increase in the αB-Cr adsorption capacity (AC0) is the main effect of trehalose on the aggregation process in the presence of the protein chaperone (AC0 increases 1.5-fold in the presence of 66 mM trehalose). According to the sedimentation analysis data, trehalose stabilizes the dimeric form of Phb at the stages of denaturation and dissociation and enhances the interaction of αB-Cr with the target protein. Moreover, trehalose shifts the equilibrium between the αB-Cr oligomers towards the smaller forms. Thus, trehalose affects the quaternary structure of αB-Cr and increases its anti-aggregation activity at the nucleation stage.

Effect of Betaine and Arginine on Interaction of αB-Crystallin with Glycogen Phosphorylase b.

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.

Combined action of chemical chaperones on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b.

Chemical chaperones are a class of small molecules, which enhance protein stability, folding, inhibit protein aggregation, and are used for long-term storage of therapeutic proteins. The combined action of chemical chaperones trehalose, betaine and lysine on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b (Phb) has been studied. Dynamic light scattering data indicate that the affinity of trehalose to Phb increased in the presence of betaine or lysine at both stages (stage of nucleation and aggregate growth) of enzyme aggregation at 48 °C, in contrast, the affinity of betaine to the enzyme in the presence of lysine remained practically unchanged. According to differential scanning calorimetry and analytical ultracentrifugation data, the mixture of trehalose and betaine stabilized Phb stronger than either of them in total. Moreover, the destabilizing effect of lysine on the enzyme was almost completely compensated by trehalose and only partially by betaine. The main protective effect of the mixtures of osmolytes and lysine is associated with their influence on the dissociation/denaturation stage, which is the rate-limiting one of Phb aggregation. Thus, a pair of chaperones affects the stability, oligomeric state, and aggregation of Phb differently than individual chaperones.

The mechanism of thermal aggregation of glutamate dehydrogenase. The effect of chemical chaperones.

Chemical chaperones are low-molecular compounds counteracting protein aggregation. Understanding of the mechanism of their effects is key to their potential use in biotechnology. The aggregation of bovine liver glutamate dehydrogenase (GDH) was studied at 40 °C and 50 °C using dynamic light scattering, analytical ultracentrifugation, size-exclusion chromatography and differential scanning calorimetry. At 40 °C the GDH aggregation proceeds through the slow stages of hexamer dissociation and formation of small oligomeric aggregates. At 50 °C these stages are transient. The rate-limiting stage of the overall aggregation process is unfolding of the protein molecule; the order of aggregation with respect to protein, n = 1. The test system based on GDH aggregation at 50 °C was used to quantify the anti-aggregation activity of chemical chaperones by comparing their half-saturation concentrations [L]0.5. Arginine ethyl ester had the highest anti-aggregation activity, with [L]0.5 = 4 ± 1 mM. For other additives, [L]0.5 was 22 ± 1 mM (arginine), 18 ± 1 mM (argininamide) and 95 ± 12 mM (proline). Arginine at concentrations up to 300 mM, argininamide at concentrations higher than 300 mM and arginine ethyl ester at concentrations higher than 500 mM enhance aggregate-aggregate sticking. These results explain the mechanism of heat-induced GDH aggregation and its peculiarities at different temperatures or in the presence of chemical chaperones.

Myopathy-associated G154S mutation causes important changes in the conformational stability, amyloidogenic properties, and chaperone-like activity of human αB-crystallin.

Glycine to serine substitution at position 154 of human αB-crystallin (αB-Cry) is behind the development of cardiomyopathy and late-onset distal myopathy. The current study was conducted with the aim to investigate the structural and functional features of the G154S mutant αB-Cry using various spectroscopic techniques and microscopic analyses. The secondary and tertiary structures of human αB-Cry were preserved mainly in the presence of G154S mutation, but the mutant protein indicated a reduced chaperone-like activity when γ-Cry as its natural partner in eye lenses was the substrate protein. Moreover, a significant reduction in the enzyme refolding ability and in vivo chaperone activity of the mutant protein were observed. Also, the mutant protein displayed reduced conformational stability upon urea-induced denaturation. Both fluorescence and electron microscopic analyses suggested that G154S mutant protein has an increased susceptibility for amyloid fibril formation. Therefore, the pathomechanism of G154S mutation can be explained by its attenuated chaperone function, decreased conformational stability, and increased amyloidogenic propensity. Some of these important changes may also alter the correct interaction of the mutated αB-Cry with its target proteins in myopathy.

The impact of concurrent training and antioxidant supplementation on the factors associated with the ocular lens opacity in diabetic rats.

The current study was aimed to investigate the protective effect of vitamins C and E (VCE) supplementation, exercise, and their concurrent application against cataract incidence in the diabetic rats. The obtained results indicated that different supplementation and training treatments were capable to preserve the lens transparency in the diabetic rats. Also, upon applying different supplementation and training treatments, the level of glutathione (GSH) and activity of antioxidant enzymes in the diabetic rats was preserved approximately close to their control levels. In addition, different treatments were capable to maintain the structural integrity of the lens proteins in diabetic rats. Moreover, VCE supplementation, exercise and their simultaneous application prevented lens crystallins of diabetic rats against fibrillation and formation of the increased oligomeric sizes. The results of this study signify the importance of antioxidant supplementation and exercise in reducing the detrimental effects of hyperglycemia on the eye lenses.

Structural and functional studies of D109A human αB-crystallin contributing to the development of cataract and cardiomyopathy diseases.

αB-crystallin (heat shock protein ß5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein (CRYAB) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A mutation has a significant impact on the important features of human αB-crystallin, including its structure, size of the protein oligomers, tendency to form amyloid fibrils, stability, and chaperone-like activity. Given the importance of aspartate 109 in maintaining the proper structure of the α-crystallin domain, its role in the dimerization and chaperone-like activity, as well as preserving protein stability through the formation of salt bridges; mutation at this important site might have critical consequences and can explain the genesis of myopathy and cataract disorders. Also, the formation of large light-scattering aggregates and disruption of the chaperone-like activity by D109A mutation might be considered as important contributing factors in development of the eye lens opacity.

The biochemical association between R157H mutation in human αB-crystallin and development of cardiomyopathy: Structural and functional analyses of the mutant protein.

In human αB-crystallin or HspB5, the substitution of arginine residue at position 157 with histidine has been reported to cause cardiomyopathy. In this study, the impact of R157H mutation on the structure, stability and functional properties of human αB-crystallin was investigated using a variety of spectroscopic techniques and microscopic analyses. Our spectroscopic analyses revealed that this mutation has a negligible impact on the secondary and tertiary structures of HspB5 but its quaternary structure underwent fundamental changes. Although the chemical stability of the mutant protein remained largely unchanged, the differential scanning calorimetry (DSC) measurement suggested that its thermal stability was reduced. As examined with transmission electron microscopy, αB-crystallin and its mutant indicated a similar tendency for the amyloid fibril formation under thermochemical stress. Dynamic light scattering (DLS) analysis suggested important changes in the quaternary (oligomeric) structures of the mutant protein as compared with the native protein counterpart. Also, the mutant protein indicated an improved chaperone-like activity under in vitro assessment. In a pH-dependent manner, the side chains of arginine and histidine have different capabilities for establishing hydrogen bonds and electrostatic interaction (salt bridge) and this variation may be sufficient to produce the larger changes that ultimately alter the interaction of this protein with other target proteins. Overall, the pathogenic contribution of this mutation in cardiomyopathy can be explained by its role in quaternary structure/stability alteration of the mutated protein.

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.

Chaperone-Like Activity of HSPB5: The Effects of Quaternary Structure Dynamics and Crowding.

Small heat-shock proteins (sHSPs) are ATP-independent molecular chaperones that interact with partially unfolded proteins, preventing their aberrant aggregation, thereby exhibiting a chaperone-like activity. Dynamics of the quaternary structure plays an important role in the chaperone-like activity of sHSPs. However, relationship between the dynamic structure of sHSPs and their chaperone-like activity remains insufficiently characterized. Many factors (temperature, ions, a target protein, crowding etc.) affect the structure and activity of sHSPs. The least studied is an effect of crowding on sHSPs activity. In this work the chaperone-like activity of HSPB5 was quantitatively characterized by dynamic light scattering using two test systems, namely test systems based on heat-induced aggregation of muscle glycogen phosphorylase b (Phb) at 48 °C and dithiothreitol-induced aggregation of α-lactalbumin at 37 °C. Analytical ultracentrifugation was used to control the oligomeric state of HSPB5 and target proteins. The possible anti-aggregation functioning of suboligomeric forms of HSPB5 is discussed. The effect of crowding on HSPB5 anti-aggregation activity was characterized using Phb as a target protein. The duration of the nucleation stage was shown to decrease with simultaneous increase in the relative rate of aggregation of Phb in the presence of HSPB5 under crowded conditions. Crowding may subtly modulate sHSPs activity.

Analysis of the data on titration of native and peroxynitrite modified αA- and αB-crystallins by Cu2+-ions.

The interaction of αA- and αB-crystallins with Cu2+ ion modulates their structure and chaperone-like activity which is important for lens transparency. Theoretical analysis of the dependences of fluorescence intensity of native αA- and αB-crystallins and αA- and αB-crystallins modified by peroxynitrite on concentration of Cu2+ ions has been carried out. It has been shown that one subunit of native αA-crystallin contains two equivalent Cu2+-binding sites. The microscopic dissociation constant for Cu2+-αA-crystallin complex (K diss) was found to be equal to 9.7 µM. For peroxynitrite modified αA-crystallin the K diss value is equal to 17 µM. One subunit of native αB-crystallin contains two non-equivalent Cu2+-binding sites. The corresponding microscopic dissociation constants for Cu2+-αB-crystallin complexes (K 1 and K 2) were found to be equal to 0.94 and 36 µM. For peroxynitrite modified αB-crystallin the K 1 and K 2 values are equal to 4.3 and 70 µM, respectively.

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.

The congenital cataract-causing mutations P20R and A171T are associated with important changes in the amyloidogenic feature, structure and chaperone-like activity of human αB-crystallin.

Cataract is the major reason for human blindness worldwide. α-Crystallin, as a key chaperone of eye lenses, keeps the lenticular tissues in its transparent state over time. In this study, cataract-causing familial mutations, P20R and A171T, were introduced in CRYАB gene. After successful expression in Escherichia coli and subsequent purification, the recombinant proteins were subjected to extensive structural and functional analyses using various spectroscopic techniques, gel electrophoresis, and electron microscopy. The results of fluorescence and Raman assessments suggest important but discreet conformational changes in human αB-Cry upon these cataractogenic mutations. Furthermore, the mutant proteins exhibited significant secondary structural alteration as revealed by FTIR and Raman spectroscopy. An increase in conformational stability was seen in the human αB-Cry bearing these congenital cataractogenic mutations. The oligomeric size distribution and chaperone-like activity of human αB-Cry were significantly altered by these mutations. The P20R mutant protein was observed to loose most of the chaperone-like activity. Finally, these cataractogenic mutant proteins exhibited an increased propensity to form the amyloid fibrils when incubated under environmental stress. Overall, the structural and functional changes in mutated human αB-Cry proteins can shed light on the pathogenic development of congenital cataracts.

Kinetic data analysis of chaperone-like activity of Wt, R69C and D109H αB-crystallins.

The α-Crystallin (α-Cry) functions as a molecular chaperone, preventing the formation of stress-induced protein aggregation which is important for maintenance of lens transparency. The kinetic data of Wt, R69C and D109H αB-Crys chaperone-like activity were obtained by UV-Vis spectroscopy in both thermal- and chemical-induced aggregation methods. The data were analyzed using physical parameters describing the aggregation process including t* (the characteristic of the stage of nucleation), and t 0.5 (the characteristic of the stage of aggregate growth) and I lim (the limiting value of the light scattering intensity). Parameter t* is duration of the lag phase and the lower t* value is associated with the higher rate of the nucleation stage. Also, the lower values of t 0.5 indicated the higher rate of aggregate growth stage. The change in parameter I lim in the presence of chaperones can be connected with the change in the size of protein aggregates. These data are related to the research article entitled "Structural and functional characterization of D109H and R69C mutant versions of human αB-crystallin: the biochemical pathomechanism underlying cataract and myopathy development" [1].

Structural and functional characterization of D109H and R69C mutant versions of human αB-crystallin: The biochemical pathomechanism underlying cataract and myopathy development.

In human αB-crystallin (αB-Cry), the highly conserved residues arginine 69 (R69) and aspartate 109 (D109) are located within a critical motif of α-crystallin domain (ACD), contributing to the subunit interactions and oligomeric assembly. Recently, two missense mutations (R69C and D109H) in human αB-Cry have been reported to cause congenital cataract and myopathy disorders. We used various spectroscopic techniques, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), gel electrophoresis and transmission electron microscopy (TEM) to show how these mutations cause significant changes in structure, amyloidogenic feature and biological function of human αB-Cry. These pathogenic mutations resulted in the important alterations of the secondary, tertiary and oligomeric (quaternary) structures of human αB-Cry. The missense mutations were also capable to significantly increase the amyloidogenic propensity of human αB-Cry and to diminish the chaperone-like activity of this protein. The above mentioned changes were observed more noticeably after D109H mutation. The detrimental effects of D109H mutation may be due to the loss of salt bridge with R120 in the dimeric interface, flagging the anti-aggregation ability of αB-Cry chaperone. In conclusion, the R69C and D109H mutations displayed a significant damaging effect on the structure and chaperone function of human αB-Cry which could be considered as their biochemical pathomechanisms in development of congenital cataract and myopathy disorders.

Characterization of arginine preventive effect on heat-induced aggregation of insulin.

Aggregation of proteins can affect their efficacy, and is especially important concerning therapeutic proteins such as insulin. Use of additives such as amino acids can counteract this deleterious process. Heat-induced aggregate formation of human insulin was kinetically studied with the use of various concentrations of the protein, at different temperatures, and in the presence of EDTA by UV-visible spectrophotometry. Effect of arginine, lysine, and histidine was then tested on the process at pH 4.8 and 45 °C. Kinetic parameters of the obtained growth curves (parameters t* and t0.5 characterizing the rate of the nucleation stage and the rate of the stage of aggregate growth respectively) were computed in all these conditions, and structure of aggregates was characterized by spectrofluorimetry, and transmission electron microscopy (TEM). Presence of high concentrations of the chelator EDTA increased aggregation. Among used additives, L-arginine (50 mM) most efficiently suppresses the heat-induced amorphous aggregation of insulin, affecting parameters t0.5 and t* presumably by preserving the protein's structure, as observed by the protein intrinsic fluorescence and CD spectra, and smaller formed aggregates in TEM images and dynamic light scattering. Docking experiment and subsequent molecular dynamics simulation indicated possible sites of interaction for arginine with the B-chain of insulin.

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.