Effects Of Osmolytes Research Articles

Frontispiece: Temperature, Hydrostatic Pressure, and Osmolyte Effects on Liquid–Liquid Phase Separation in Protein Condensates: Physical Chemistry and Biological Implications

Frontispiece: Temperature, Hydrostatic Pressure, and Osmolyte Effects on Liquid–Liquid Phase Separation in Protein Condensates: Physical Chemistry and Biological Implications

Temperature, Hydrostatic Pressure, and Osmolyte Effects on Liquid-Liquid Phase Separation in Protein Condensates: Physical Chemistry and Biological Implications.

Liquid-liquid phase separation (LLPS) of proteins and other biomolecules play a critical role in the organization of extracellular materials and membrane-less compartmentalization of intra-organismal spaces through the formation of condensates. Structural properties of such mesoscopic droplet-like states were studied by spectroscopy, microscopy, and other biophysical techniques. The temperature dependence of biomolecular LLPS has been studied extensively, indicating that phase-separated condensed states of proteins can be stabilized or destabilized by increasing temperature. In contrast, the physical and biological significance of hydrostatic pressure on LLPS is less appreciated. Summarized here are recent investigations of protein LLPS under pressures up to the kbar-regime. Strikingly, for the cases studied thus far, LLPSs of both globular proteins and intrinsically disordered proteins/regions are typically more sensitive to pressure than the folding of proteins, suggesting that organisms inhabiting the deep sea and sub-seafloor sediments, under pressures up to 1 kbar and beyond, have to mitigate this pressure-sensitivity to avoid unwanted destabilization of their functional biomolecular condensates. Interestingly, we found that trimethylamine-N-oxide (TMAO), an osmolyte upregulated in deep-sea fish, can significantly stabilize protein droplets under pressure, pointing to another adaptive advantage for increased TMAO concentrations in deep-sea organisms besides the osmolyte's stabilizing effect against protein unfolding. As life on Earth might have originated in the deep sea, pressure-dependent LLPS is pertinent to questions regarding prebiotic proto-cells. Herein, we offer a conceptual framework for rationalizing the recent experimental findings and present an outline of the basic thermodynamics of temperature-, pressure-, and osmolyte-dependent LLPS as well as a molecular-level statistical mechanics picture in terms of solvent-mediated interactions and void volumes.

Common Source of Cryoprotection and Osmoprotection by Osmolytes.

While recent studies clarify the effect of osmolytes on Coulomb interaction at elevated concentrations of salt, little is known about the way osmolytes affect the same interaction in cryoprotection. In this Communication we explore the effect of cold on the interaction between two charged surfaces immersed in ternary solution containing salt and osmolyte and find that the effect of cold parallels that of excess salt, i.e., low temperatures increase adsorption of salt counterions to the surface, thus neutralizing it. Two osmolytes, proline and glycine-betaine, are then shown to recharge the surface by releasing the adsorbed counterions. The ability to counteract effects of both cold and excess salt on Coulomb interactions renders these known osmolytes cryoprotectants as well as osmoprotectants, explaining why plants, fish, insects and bacteria accumulate them in response to either drought or cold stress.

Counteracting Effect of Charged Amino Acids Against the Destabilization of Proteins by Arginine.

Studies on osmolyte-induced effects on proteins help in enhancing protein stability under stressed conditions for various applications. Using mixtures of osmolytes could indeed widen their applications. The combinatorial effects of osmolytes with methylamines are majorly found in the literature; however, such studies are limited on the amino acid class of osmolytes. The present study examines the effect of charged amino acids Arg, Asp, and Lys on the stability of RNase A and α-LA. The thermal stabilities of the proteins in the presence of osmolytes are monitored by absorption changes, and the structural changes are analyzed using fluorescence quenching and near-UV circular dichroism (CD). These results are compared with our previous report on the effect of Glu. Arg destabilizes both the proteins whereas Asp, Lys, and Glu stabilize the proteins. The extent of stability provided by Asp and Glu is almost same and higher than Lys in RNase A. However, the stability acquired in the presence of Asp and Lys is comparable for α-LA and Glu provides higher stability. Further, the quenching and CD results suggest that the addition of amino acids do not alter the structure of the proteins significantly. The counteracting abilities of the stabilizing amino acids (stAAs) against Arg are then investigated. The results show that Glu could counteract Arg at the lowest fraction in the mixture. Lys requires nearly equimolar concentration whereas Asp needs almost double the concentration to counteract Arg induced destabilization of the proteins. At higher concentrations, the counteracting ability of Asp and Lys is similar for both the proteins. The counteracting ratio might slightly vary among the proteins, and it is not necessary that the amino acid providing higher stability to the protein could more effectively counteract Arg. This could be due to the change in the extent of preferential hydration of the proteins by stAAs in the presence of Arg. The results suggest that the addition of stAAs could be an effective strategy to increase the protein stability in biotechnology and biopharma applications.

Ligand binding studies of a trimethoprim‐resistant dihydrofolate reductase by fluorine NMR

Antibiotic resistance is a worldwide problem. The excessive use and misuse of antibiotics have resulted in the development of drug resistance. One example involves the antibiotic trimethoprim (TMP). TMP selectively inhibits the bacterial enzyme dihydrofolate reductase (DHFR) over its human counterpart. Inhibition of DHFR results in inhibition of DNA synthesis and ultimately in cell death. Unfortunately, extensive use and dissemination of TMP over the last forty years has resulted in the development of drug resistance in the form of highly transmissible plasmids. R67 dihydrofolate reductase is a plasmid‐encoded enzyme that confer resistance to TMP. Our lab has studied how R67 DHFR works in vitro using diluted conditions. However, most proteins function inside cells under crowded and heterogeneous environment. As 19F labeled proteins in complex mixtures can be characterized by NMR, we report our initial expression and characterization of R67 DHFR. To obtain such information, we have labeled R67 DHFR with fluorine at different positions in the indole ring and showed that fluorine incorporation into R67 DHFR does not affect the structure and function of the protein. We have characterized the 19F NMR spectra of apo R67 DHFR and optimized fluorine incorporation. Furthermore, fluorinated R67 DHFR shows different signals for binding of cofactor, NADP+, and substrate, dihydrofolate (DHF). We have obtained similar dissociation constants for the cofactor and substrate of R67 DHFR by both NMR and ITC. In our next step, we will measure dissociation constants in more complex solutions. We envision using fluorinated protein to quantify the effect of osmolytes and crowders on the binding affinity of substrate and cofactor towards R67 DHFR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of ectoine, hydroxyectoine and β-hydroxybutyrate on the temperature and pressure stability of phospholipid bilayer membranes of different complexity.

Previous research has shown that ectoines fluidize lipid monolayers by increasing the liquid expanded region in DPPC monolayers and also decreasing the line tension responsible for the phase morphology. Here, we explored possible effects of the compatible osmolytes ectoine, hydroxyectoine and β-hydroxybutyrate on lipid bilayer membranes, including effects of temperature and pressure. The effect of the protective osmolytes on the phase transition of DPPC bilayers was investigated by fluorescence spectroscopy, differential scanning calorimetry and pressure perturbation calorimetry. A slight change of the phase behavior was observed, which resulted in a stabilization of the gel phase, which may be caused by an alteration of the hydration properties at the lipid interface and H-bond and electrostatic interactions in the headgroup region. We then explored the cosolvents' effects on giant unilamellar vesicles (GUVs) formed by lipid mixtures exhibiting phase separation into liquid-ordered (lo) and liquid-disordered (ld) domains using BODIPY-PC and the DiI18 dye as labels. The presence of both, ectoine and hydroxyectoine showed significant effects on the lateral organization increasing the fluid domains. Moreover, we observed a considerable increase in the adhesion behavior of small vesicles onto GUV surfaces. Diffusion studies by fluorescence recovery after photobleaching experiments on POPC giant vesicles quantitatively showed a hydroxyectoine-induced increase of the diffusion coefficient values, clearly demonstrating an increase in the lateral mobility of lipid within the bilayer membrane. This study provides clear evidence for the fluidizing effect of the compatible solutes on bilayer lipid membranes. A marked effect, however, was only detected if phase separated domains exist.

Effect of osmolytes on the activity of anti-cancer enzyme L-Asparaginase II from Erwinia chrysanthemi

Abstract L-asparaginase is used for the treatment of acute lymphoblastic leukaemia (ALL); however, its formulation presents drawbacks such as a lack of stability and formation of aggregates. Osmolytes are small molecules accumulated by cells in response to environmental stresses and present a protective behaviour, favouring the equilibrium of macromolecules towards the native conformation. Therefore, osmolytes are employed as excipients in pharmaceutical protein formulations. Herein, recombinant L-ASNase II from Erwinia chrysanthemi (ErA II) was analysed with respect to the effect of osmolytes on kinetic and stability of this biopharmaceutical. The aggregation profiles were analysed trough nanotracking particle analysis and dynamic light scattering. The majority of the tested osmolytes increased ErA II specific activity and stability, being more pronounced for sucrose and sorbitol, which increased almost 70% of ErA II activity. The polyol preserved total enzyme activity for 30 days while sucrose preserved 81.1 ± 5.3% total enzyme activity over this period. Each osmolyte resulted in a specific aggregation profile and the presence of sucrose or sorbitol resulted in a lower quantity of aggregates in the range of 100–300 nm. The present findings may contribute to the improvement of adjuvants in L-ASNase formulations and the optimization of other biopharmaceutical formulations.

Equilibration of molecules between two compartments through a nanochannel in the presence of osmolytes: a molecular dynamics simulation study.

Using molecular dynamics simulations, we study the equilibration of a system consisting of two nanoscale compartments connected by a carbon nanotube through which small mobile molecules can pass. The system is initially in a state where only one compartment is filled with molecules and the other is empty. When the molecules are allowed to move from the filled compartment to the empty one, the system starts equilibrating and finally reaches an equilibrium state where the molecules are distributed between the two compartments. In the absence of osmolytes, the equilibrium distribution of molecules is simply determined by the relative volumes of the compartments, but in the presence of osmolytes, the distribution is dependent on not only the relative compartment volumes but also the osmolyte properties. To systematically study the effect of osmolytes, we investigate how the number of osmolytes and the strength of the interaction between molecules and osmolytes affect the equilibrium state. Interestingly, we find that osmolytes strongly interacting with molecules can drain the initially filled compartment and induce the complete transfer of molecules to the initially empty compartment. We also study the kinetic and thermodynamic aspects of the equilibration processes.

Do Osmolytes Impact the Structure and Dynamics of Myoglobin?

Osmolytes are small organic compounds that can affect the stability of proteins in living cells. The mechanism of osmolytes’ protective effects on protein structure and dynamics has not been fully explained, but in general, two possibilities have been suggested and examined: a direct interaction of osmolytes with proteins (water replacement hypothesis), and an indirect interaction (vitrification hypothesis). Here, to investigate these two possible mechanisms, we studied myoglobin-osmolyte systems using FTIR, UV-vis, CD, and femtosecond IR pump-probe spectroscopy. Interestingly, noticeable changes are observed in both the lifetime of the CO stretch of CO-bound myoglobin and the spectra of UV-vis, CD, and FTIR upon addition of the osmolytes. In addition, the temperature-dependent CD studies reveal that the protein’s thermal stability depends on molecular structure, hydrogen-bonding ability, and size of osmolytes. We anticipate that the present experimental results provide important clues about the complicated and intricate mechanism of osmolyte effects on protein structure and dynamics in a crowded cellular environment.

Zwitterionic Osmolytes Resurrect Electrostatic Interactions Screened by Salt.

Many cells synthesize significant quantities of zwitterionic osmolytes to cope with the osmotic stress induced by excess salt. In addition to their primary role in balancing osmotic pressure, these osmolytes also help stabilize protein structure and restore enzymatic activity compromised by high ionic strength. This osmoprotective effect has been studied extensively, but its electrostatic aspects have somehow escaped the mainstream. Here, we report that, despite their overall neutrality, zwitterions may dramatically affect electrostatic interactions in saline solutions of biological relevance. Using atomic force microscopy, we study the combined effect of osmolytes and salts on electrostatic interactions between two negatively charged silica surfaces in mixtures of five salts (NaCl, KCl, CsCl, MgCl2, and CaCl2) and five zwitterionic osmolytes (betaine, proline, trimethylamine N-oxide, glycine, and sarcosine) as a function of solutes concentration and pH. All osmolytes are found to counteract the screening effect of salt on electrostatic repulsion, albeit to a different extent. They do so by both increasing the screening length shortened by added salts and by desorbing bound protons and cations, hence enhancing the negative surface charge. Both effects are traced to the osmolytes' higher molecular polarizability compared with water. In addition to this direct effect on the solution's dielectric constant, we identify an osmolytic Hofmeister effect with the more hydrophobic osmolytes more efficiently desorbing weakly hydrated cations from weakly hydrated silica and the less hydrophobic osmolytes better desorbing strongly hydrated cations from strongly hydrated silica. The combined results shed light on Coulomb interactions in a more realistic model of the cytosol, a relatively unexplored territory.

Effect of osmolytes of different type on DNA behavior in aqueous solution. Experimental and theoretical studies

Osmolytes, the small organic molecules accumulated in cells under environmental stress, can modulate the stability of biopolymers such as proteins and DNA. In spite of many years of research, there is no established molecular mechanism of the influence of osmolytes on DNA structure. Here, we used two model osmolytes that denature (urea) or stabilize (trimethylglycine, TMG) proteins to study their effect on DNA in aqueous solutions using a combination of spectroscopic, calorimetric and simulation methods. We found that both urea and TMG dehydrate DNA and shift the conformational equilibrium from B- to A-DNA form, despite different solvation properties. The isolated vibrational spectra of osmolytes and analysis of preferential accumulation indicate a lack of direct specific interaction with DNA in ternary solutions. Besides the influence of TMG and urea on DNA hydration, we studied and analyzed the orientational and spatial distribution of osmolytes around DNA. Analysis of hydrogen bonds and theoretical calculations of interactions with native and denatured DNA show strong negative interactions with bases of dissociated DNA: while TMG preferentially interacts with guanine, urea interacts equally strongly with all bases. This high affinity of both TMG and urea towards DNA bases appears to be the cause of their denaturing properties.

Influence and effect of osmolytes in biopharmaceutical formulations

Osmolytes are small organic molecules accumulated by cells in response to environmental stresses. They are represented by amino acids, sugars, polyols, tertiary sulphonium and quaternary ammonium compounds. These molecules present a protective behaviour and favour the equilibrium of macromolecules towards the native form, preventing denaturation and promoting the folding of unfolded proteins. Protein formulations due to their biological character require greater care during the manufacturing process, shelf-life and administration of the drug, as variations in these factors may result in denaturation, inactivation and/or protein aggregation. These drawbacks can be surpassed using osmolytes as excipients in protein formulations as stabilisers, bulking agents and even buffers. A number of 133 biologics, including vaccines and immunoglobulins, approved by the U.S. Food and Drug Administration (FDA) between 1998 and 2017 were analysed in this work in order to identify the most used group of osmolyte molecules. A deep insight into their role in protein formulations was discussed and compared to data in the literature. The advantages and disadvantages of their use in specific formulations were also extensively discussed here. In conclusion, investigation into the role of osmolytes in each formulation is essential for understanding their effect and provides a background to be used when selecting the best osmolyte to fit a specific formulation without excluding the patient needs.

Protective Effect of Biological Osmolytes against Heat- and Chaotropic Agent-Induced Denaturation of Bacillus licheniformis γ-Glutamyl Transpeptidase.

In the present study, the stabilizing effect of four different biological osmolytes on Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT) was investigated. BlGGT appeared to be stable under temperatures below 40°C, but the enzyme retained less than 10% of its activity at 60°C. The tested osmolytes exhibited different degrees of effectiveness against temperature inactivation of BlGGT, and sucrose was found to be the most effective among these. The use of circular dichroism spectroscopy for studying the secondary structure of BlGGT revealed that the temperature-induced conformational change of the protein molecule could be prevented by the osmolytes. Consistently, the molecular structure of the enzyme was essentially conserved by the osmolytes at elevated temperatures as monitored by fluorescence spectroscopy. Sucrose was further observed to counteract guanidine hydrochloride (GdnHCl)- and urea-induced denaturation of BlGGT. Taken together, we observed evidently that some well-known biological osmolytes, especially sucrose, make a dominant contribution to the structural stabilization of BlGTT.

Effect of polyols on the structure and aggregation of recombinant human γ-Synuclein, an intrinsically disordered protein

Polyol osmolytes accumulated in cells under stress are known to promote stability in globular proteins with respect to their increasing hydroxyl groups but their effect on the structure, stability and aggregation of intrinsically disordered proteins (IDPs) is still elusive. The lack of a natively folded structure in intrinsically disordered proteins under physiological conditions results in their aggregation and fibrillation that gives rise to a number of diseases. We have investigated the effect of a series of polyols, ethylene glycol (EG), glycerol, erythritol, xylitol and sorbitol on the fibrillation pathway of recombinant human γ-Synuclein, used as a model, for an IDP known to form fibrils that play a role in neurodegeneration and cancer. With an increase in the number of –OH groups in polyols except EG, we observe a decrease in lag time for fibrillation at equimolar concentrations, suggesting stronger preferential exclusion of polyols that promotes γ-Syn self-association and oligomerization. The polyols act early during nucleation and their diverse effect on the rate of fibrillation suggests the role of favourable solvent-side chain interactions. With increasing –OH group, polyols stabilize the natively unfolded conformation of γ-Syn under non-fibrillating conditions and delay the structural transition to characteristic β-sheet structure by forming an α-helical intermediate during fibrillation. The results, overall suggest that the effect of osmolytes on IDPs is much more complex than their effect on globular protein stability and aggregation and a fine balance between the dominant unfavourable osmolyte-peptide backbone and favourable osmolyte-charged side chain interactions would govern their stability and aggregation properties.

Osmolytes and Protein-Protein Interactions.

Cells survive fluctuations in osmolality by accumulating and depleting highly soluble, usually neutral, small organic compounds. Natural selection has converged on a small set of such molecules, called osmolytes. The biophysical characterization of osmolytes, with respect to proteins, has centered on tertiary structure stability. Data about their effect on protein assemblies, whose formation is driven by surface interactions, is lacking. Here, we investigate the effects of osmolytes and related molecules on the stabilities of a protein and a protein complex. The results demonstrate that osmolytes are not differentiated from other cosolutes by their stabilizing influences on protein tertiary structure but by their compatibility with the interactions between protein surfaces that organize the cellular interior.

A comparative study of structure, stability and function of sc-tenecteplase in the presence of stabilizing osmolytes

The aim of the present study was to investigate the effect of three routine drug excipients, as osmolytes, in three different concentrations, on structure, thermal stability and the activity of single-chain (sc-) tenecteplase. To see the influence of trehalose, mannitol, and sucrose on the structure, stability and function of sc-tenecteplase, thermal stability, fluorescence, circular dichroism (CD) and enzyme kinetic measurements and molecular docking studies were carried out. To measure the effect of osmolytes on stability of sc-tenecteplase, thermodynamic parameters (transition temperature (Tm), standard enthalpy change (ΔH°), standard entropy change (ΔS°) and ΔG°, the standard Gibbs free energy change, were determined from heat-induced transition curves of the protein in absence and presence of each osmolyte. It was observed that all three osmolytes acted as an enhancer for the sc-tenecteplase stability, with varying efficacies and efficiencies. The results of the kinetic study showed that the activity of sc-tenecteplase is increased in the presence of osmolytes. The near-UV and far-UV CD studies showed transfer of Trp, Phe and Tyr residues to a more flexible environment in the presence of osmolytes. The sc-tenecteplase fluorescence quenching suggested the more polar location of Trp residues. Molecular docking studies revealed that (i) Gibbs free energy of interaction between the osmolyte and sc-tenecteplase is negative, and (ii) hydrogen bond and hydrophobic interactions dominate within the interaction sites.

Sugar osmolytes-induced stabilization of RNase A in macromolecular crowded cellular environment

Organisms synthesize sugar osmolytes during environmental stresses to protect proteins against denaturation. These studies were carried out in dilute buffer whereas intracellular milieu within cells has cytoplasmic concentration of macromolecules in the range of 80–400 mg ml−1. Is the stabilizing effect of sugar osmolytes on the protein in dilute buffer different from that when protein is in cellular environment? To answer this question, we have measured and analysed the effect of sugar osmolytes on the structural and thermodynamic stability of ribonuclease A in the presence of dextran 70 at multiple concentrations of six sugars at different pH values. It was found that (i) each sugar osmolyte in the crowded environment provides stability to the protein in terms of Tm (midpoint of denaturation) and ∆GD° (Gibbs energy change) and this stabilizing effect is under entropic control, (ii) the extent of osmolyte-induced stabilization of RNase A is pH dependent, and (iii) effect of sugars on the stability of protein in presence of the crowding agent remains unchanged. This study concludes that crowding does not affect the efficacy of osmolytes and vice versa; and emphasizes on understanding of internal architecture of the cellular environment with respect to molecular and macromolecular crowding.

Effect of osmotic stress on folate pathway enzyme.

How enzymes behave in cells may differ from how they behave in the test tube. Previous in vitro studies of three different dihydrofolate reductases as well as biophysical studies of folate find that osmolytes interact weakly with folate and dihydrofolate thereby replacing water in the hydration shell at certain functional groups. Removal of the osmolyte from the solvation shell of dihydrofolate is more difficult than removal of water, which weakens the Kd value. To determine if this phenomenon occurs in vivo, osmotic stress titrations of several folate pathway enzymes were performed in E. coli. Knockout and rescued strains of folate pathway enzymes were made in E. coli and subjected to osmotic stress. The bacterium synthesizes osmolytes in response to osmotic stress. Comparison of the growth of the knockout and rescued strains of the folate pathway enzymes would give us a measure of the effect of osmotic stress on the particular enzyme activity. We have found that the knockout strain could grow to high osmolalities on supplemented media while the rescued strain stopped growing at lower osmolalities on minimal media. This growth pattern was observed for an R67 dihydrofolate clone rescuing a ΔfolA strain of E. coli, for a methylenetetrahydrofolate reductase clone rescuing a Keio ΔmetF strain and for a serine hydroxymethyl transferase clone rescuing a Keio ΔglyA strain. While many parameters may be involved in these osmotic stress titrations, math modeling of the folate pathway suggests that these results could be an effect of direct titration of the enzyme activity or may be due to domino/cascading effects. We have further explored the effect of osmotic stress on E. coli DH5α strain with the dihydropteroate synthase (DHPS) gene in a tunable plasmid. The ability of the enzyme to function and rescue DH5α cells in the presence of sulfamethoxazole was determined. This was done to study the effect of osmotic stress on the functioning of DHPS with respect to growth of the cells in minimum inhibitory concentrations of the drug. From our previous osmotic stress titrations, the in vivo enzyme activity can be titrated directly by osmotic stress or can be inhibited due to domino effects. We are studying the effects of osmolytes to DHPS by using Isothermal Titration Calorimetry. Thus, by combining in vitro and in vivo studies, we will be able to answer if the activity of DHPS is affected in vivo. This will help to bridge the gap between in vivo and in vitro studies with enzymes. Model of osmolyte interaction with DHF that results in weaker binding to DHFR. For enzyme assays in buffer, DHF binds tightly to DHFR (target enzyme) and water (blue) is released. Added osmolytes (magenta spheres) interact weakly with DHF. For DHF to bind to DHFR, both osmolytes and water need to be released. While these interactions are weak, if the osmolyte-DHF interaction is stronger than the water-DHF interaction, then binding to DHFR is weakened. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thermodynamics of Concanavalin A Self-Association in the Presence of Osmolytes

Protein-protein interactions are critical for biological function and depend significantly on environmental factors. A wide variety of small organic molecules that comprise the cellular environment are capable of interacting with proteins to affect folding, binding, and association. The plant lectin concanavalin A (ConA) undergoes a reversible, pH dependent dimer-to-tetramer equilibrium and has been used in our laboratory as a model system to study the effect of osmolytes on protein self-association. Previous research determined that trimethylamine N-oxide (TMAO) stabilizes the tetrameric conformation, while urea favors the dimer. Studying the equilibrium over a range of temperatures allowed quantification of the enthalpy change (∆H) and entropy change (∆S) of tetramer formation. Urea increased both the ∆H and ∆S of tetramer formation, while TMAO decreased both. These effects are consistent with preferential hydration of the dimer-dimer interface in TMAO solution and preferential binding of urea to the interface. KEYWORDS: Concanavalin A; Osmolytes; Trimethylamine N-oxide; Urea; Protein-Protein Interaction; Equilibrium; Enthalpy; Preferential Hydration

Effects of osmolytes on the refolding of recombinant Pelodiscus sinensis brain-type creatine kinase

Abstract The protective effects of osmolytes on the renaturation of Pelodiscus sinensis brain-type creatine kinase (P-CKB) were determined in this study. The P-CKB gene was cloned and was heterologously expressed in Escherichia coli BL21 (DE3). The purified recombinant protein was subjected to 6 M urea denaturation and further applied with six different osmolytes (glycine, proline, sorbitol, DMSO, betaine, and trehalose). Our results demonstrated that the addition of glycine or DMSO could contribute toward the reactivation of unfolded P-CKB and prevent aggregation. Interestingly, 25 mM glycine was found to be the best concentration to reactivate denatured P-CKB, while high-concentration glycine led to the opposite effect as monitored by a red shift in the intrinsic fluorescence spectra and the aggregation of protein, which probably could be attributed to an additional inactive protein complex that formed during the fast refolding reaction. Docking simulation results showed the osmolyte docking energies to be relatively low and clustering groups were spread on the surface of P-CKB, indicating that osmolytes directly protected the surface of P-CKB. The results in this study could provide a better understanding of structural and functional changes in P-CKB during refolding in addition to the role of osmolytes in heterothermic animals.