Presence Of Osmolytes Research Articles

Protein Folding in the Presence of Osmolytes - a Complex Interplay of Multiple Forces

Protein Folding in the Presence of Osmolytes - a Complex Interplay of Multiple Forces

Effects of osmolytes under crowding conditions on the properties of muscle glycogen phosphorylase b

The study of the relationship between the activity and stability of enzymes under crowding conditions in the presence of osmolytes is important for understanding the functioning of a living cell. The effect of osmolytes (trehalose and betaine) on the secondary and tertiary structure and activity of muscle glycogen phosphorylase b (Phb) under crowding conditions created by PEG 2000 and PEG 20000 was investigated using dynamic light scattering, differential scanning calorimetry, circular dichroism spectroscopy, fluorimetry and enzymatic activity assay. At 25 °C PEGs increased Phb activity, but PEG 20000 to a greater extent. Wherein, PEG 20000 significantly destabilized its tertiary and secondary structure, in contrast to PEG 2000. Trehalose removed the effects of PEGs on Phb, while betaine significantly reduced the activating effect of PEG 20000 without affecting the action of PEG 2000. Under heat stress at 48 °C, the protective effect of osmolytes under crowding conditions was more pronounced than at room temperature, and the Phb activity in the presence of osmolytes was higher in these conditions than in diluted solutions. These results provide important insights into the complex mechanism, by which osmolytes affect the structure and activity of Phb under crowding conditions.

Amyloid Aggregation Is Potently Slowed Down by Osmolytes Due to Compaction of Partially Folded State

Amyloid aggregation is a key process in amyloidoses and neurodegenerative diseases. Hydrophobicity is one of the major driving forces for this type of aggregation, as an increase in hydrophobicity generally correlates with aggregation susceptibility and rate. However, most experimental systems in vitro and prediction tools in silico neglect the contribution of protective osmolytes present in the cellular environment. Here, we assessed the role of hydrophobic mutations in amyloid aggregation in the presence of osmolytes. To achieve this goal, we used the model protein human muscle acylphosphatase (mAcP) and mutations to leucine that increased its hydrophobicity without affecting its thermodynamic stability. Osmolytes significantly slowed down the aggregation kinetics of the hydrophobic mutants, with an effect larger than that observed on the wild-type protein. The effect increased as the mutation site was closer to the middle of the protein sequence. We propose that the preferential exclusion of osmolytes from mutation-introduced hydrophobic side-chains quenches the aggregation potential of the ensemble of partially unfolded states of the protein by inducing its compaction and inhibiting its self-assembly with other proteins. Our results suggest that including the effect of the cellular environment in experimental setups and predictive softwares, for both mechanistic studies and drug design, is essential in order to obtain a more complete combination of the driving forces of amyloid aggregation.

Escherichia coli O157:H7 had a high degree of acid resistance in the presence of osmolytes (glycerol, glycine or fructose) by altering its lipid membrane composition

This study aims to investigate the resistance of E. coli O157:H7 to acetic acid (AA) or malic acid (MA) by adding osmolytes, such as glycerol, glycine, glucose, and fructose, in Luria-Bertani broth without NaCl (LBW/S) or phosphate buffer (PB) stored at 25 °C. In LBW/S, a significantly (p < 0.05) higher D-value of E. coli O157:H7 was observed when treated with AA and 20% glycine (D-value: 1.18–3.44) or 40% glucose (D-value: 1.05–2.52) compared to that of AA alone (D-value: 0.40–0.47). In contrast, the addition of osmolytes (i.e. 3-40% glucose, 3–40% fructose or 20% glycine) to LBW/S acidified by MA significantly decreased D-values of E. coli O157:H7, which was enumerated by using a selective medium. Furthermore, when E. coli O157:H7 was incubated in LBW/S containing AA and osmolytes at 25 °C for 3 d, this bacterium had an increased proportion of C16:0 and C17:0 cyclo (cyclopropane acid) compared to its AA-treated counterparts. Along with the altered shift in membrane phospholipids, the addition of osmolytes into a laboratory medium in the presence of nutritive substrates may increase the resistance of E. coli O157:H7 to AA.

Insights into the dynamic interactions of RNase a and osmolytes through computational approaches

Osmolytes are small organic molecules that are known to stabilize proteins and other biological macromolecules under various stressful conditions. They belong to various categories such as amino acids, methylamines, and polyols. These substances are commonly known as ‘compatible solutes’ because they do not disrupt cellular processes and help regulate the osmotic balance within cells. In the case of ribonuclease A (RNase A), which is prone to aggregation, the presence of osmolytes can help to maintain its structural stability and prevent unwanted interactions leading to protein aggregation. In this study, we investigated the interaction between RNase A and several osmolytes using molecular docking and molecular dynamics (MD) simulations. We performed molecular docking to predict the binding mode and binding affinity of each osmolyte with RNase A. MD simulations were then carried out to investigate the dynamics and stability of the RNase A-osmolyte complexes. Our results show that two osmolytes, glucosylglycerol and sucrose have favorable binding affinities with RNase A. The possible role of these osmolytes in stabilizing the RNase A and prevention of aggregation is also explored. By providing computational insights into the interaction between RNase A and osmolytes, the study offers valuable information that could aid in comprehending the mechanisms by which osmolytes protect proteins and help in designing therapeutics for protein-related disorders based on osmolytes. These findings may have significant implications for the development of novel strategies aimed at preventing protein misfolding and aggregation in diverse disease conditions. Communicated by Ramaswamy H. Sarma

Effect of osmolytes on the EcoRI endonuclease: Insights into hydration and protein dynamics from molecular dynamics simulations

Osmolytes play an important role in cellular physiology by modulating the properties of proteins, including their molecular specificity. EcoRI is a model restriction enzyme whose specificity to DNA is altered in the presence of osmolytes. Here, we investigate the effect of two different osmolytes, glycerol and DMSO, on the dynamics and hydration of the EcoRI enzyme using molecular dynamics simulations. Our results show that the osmolytes, alter the essential dynamics of EcoRI. Particularly, we observe that the dynamics of the arm region of EcoRI which is involved in DNA binding is significantly altered. In addition, conformational free energy analyses reveals that the osmolytes bring about a change in the landscape similar to that of EcoRI bound to cognate DNA. We further observe that the hydration of the enzyme for each of the osmolyte is different, indicating that the mechanism of action of each of these osmolytes could be different. Further analyses of interfacial water dynamics using rotational autocorrelation function reveals that while the protein surface contributes to a slower tumbling motion of water, osmolytes, additionally contribute to the slowing of the angular motion of the water molecules. Entropy analysis also corroborates with this finding. We also find that the slowed rotational motion of interfacial waters in the presence of osmolytes contributes to a slowed relaxation of the hydrogen bonds between the interfacial waters and the functionally important residues in the protein. Taken together, our results show that osmolytes alter the dynamics of the protein by altering the dynamics of water. This altered dynamics, mediated by the changes in the water dynamics and hydrogen bonds with functionally important residues, may contribute to the altered specificity of EcoRI in the presence of osmolytes.

Interaction of lectin Sambucus nigra with sialylated trisaccharides in the presence of osmolytes. Chronopotentiometric sensing

Trisaccharides bind to their interaction partners-lectins relatively weakly, which makes detection of their complexes challenging. In this work, we show that an osmolyte presence improves the distinguishing complexes of lectin Sambucus nigra with trisialyllactoses with various binding affinities. The addition of osmolyte, non-binding sugar mannose significantly improved the precision of binding experiments performed using chronopotentiometric stripping at the electrode surface and fluorescence analysis in solution. Osmolytes minimized nonspecific interactions between binding sugar and lectin. Obtained findings can be utilized in any in vitro methods studying interactions of carbohydrates, respectively their conjugates with proteins. The study of carbohydrate interactions appears important since they play essential roles in a variety of biological processes including carcinogenesis.

Methanol and Sorbitol Affect the Molecular Dynamics of Arginine Deiminase: Insights for Improving its Stability

Background: Arginine deiminase enzyme of Mycoplasma arginini (MaADI) is a potential anti-cancer agent for treating arginine-auxotrophic cancers. Investigating the protein stability in the presence of osmolytes can help to increase protein stability under various stressed conditions. Methods: In this study, the stability and dynamics of MaADI were investigated in pure water and solutions of 1 M sorbitol, 10% (v/v) methanol, and 50% (v/v) methanol using molecular dynamics simulation. Results: Sorbitol was found to stabilize the protein, whereas high-concentrated methanol destabilized it. Sorbitol molecules interacted with the protein through hydrogen bonding and reduced the protein fluctuations as well. At 50% methanol, the flexibility of regions 4-8, 195-201, 314-324, and 332-337 in the MaADI was increased; whereas residues 195-201 showed the highest variations. Conclusion: Thus, these regions of MaADI, especially 195-201, are the most sensitive regions in the presence of denaturing agents and can be subjected to protein engineering toward improving the stability of MaADI.

In silico studies of the human IAPP in the presence of osmolytes

The human islet amyloid polypeptide or amylin is secreted along with insulin by pancreatic islets. Under the drastic environmental conditions, amylin can aggregate to form amyloid fibrils. This amyloid plaque of hIAPP in the pancreatic cells is the cause of type II diabetes. Early stages of amylin aggregates are more cytotoxic than the matured fibrils. Here, we have used the all-atom molecular dynamic simulation to see the effect of water, TMAO, urea and urea/TMAO having ratio 2:1 of different concentrations on the amylin protein. Our study suggest that the amylin protein forms β-sheets in its monomeric form and may cause the aggregation of protein through the residue 13-17 and the C-terminal region. α-Helical content of protein increases with an increase in TMAO concentration by decreasing the SASA value of protein, increase in intramolecular hydrogen bonds and on making the short-range hydrophobic interactions. Electrostatic potential surfaces show that hydrophobic groups are buried and normalised configurational entropy of backbone, and side-chain atoms is lesser in the presence of TMAO, whereas opposite behaviour is obtained in the case of urea. Counteraction effect of TMAO using Kast model towards urea is also observed in ternary solution of urea/TMAO.

Response to crowded conditions reveals compact nucleus for amyloid formation of folded protein.

Although the consequences of the crowded cell environments may affect protein folding, function and misfolding reactions, these processes are often studied in dilute solutions in vitro. We here used biophysical experiments to investigate the amyloid fibril formation process of the fish protein apo-β-parvalbumin in solvent conditions that mimic steric and solvation aspects of the in vivo milieu. Apo-β-parvalbumin is a folded protein that readily adopts an amyloid state via a nucleation-elongation mechanism. Aggregation experiments in the presence of macromolecular crowding agents (probing excluded volume, entropic effects) as well as small molecule osmolytes (probing solvation, enthalpic effects) revealed that both types of agents accelerate overall amyloid formation, but the elongation step was faster with macromolecular crowding agents but slower in the presence of osmolytes. The observations can be explained by the steric effects of excluded volume favoring assembled states and that amyloid nucleation does not involve monomer unfolding. In contrast, the solvation effects due to osmolyte presence promote nucleation but not elongation. Therefore, the amyloid-competent nuclei must be compact with less osmolytes excluded from the surface than either the folded monomers or amyloid fibers. We conclude that, in contrast to other amyloidogenic folded proteins, amyloid formation of apo-β-parvalbumin is accelerated by crowded cell-like conditions due to a nucleation process that does not involve large-scale protein unfolding.

An improved strategy of TGFβ3 expression in Escherichia coli: Exploiting folding modulators for a switch from misfolded to folded form

Transforming growth factor beta 3 (TGFβ3) exhibits a complex native structure featuring the presence of multiple disulfide bonds forming the active dimer. Consequently, its heterologous expression in microbial system invariably leads to inclusion body (IB) formation. In this study, we observed an interesting phenomenon of switching a significant fraction of misfolded TGFβ3 to folded form by modulating the cellular protein folding machinery. We carried out co-expression experiments with chaperones and demonstrated the requirement of a coordinated action of DnaK-DnaJ-GrpE and GroESL, to achieve the native soluble conformation of TGFβ3, during over-expression in E. coli. The novelty of this study lies in the fact that orchestration of a group of chaperones to work in concert for efficient folding and assembly of TGFβ3-like cytokines has not been widely explored. Additionally, we have also demonstrated that presence of osmolytes (sorbitol or trehalose) in the growth media have an appreciable impact on the solubility of TGFβ3. We have further shown a synergism between the effects of molecular chaperone and osmolytes on the solubility of TGFβ3. We have confirmed the functionality of soluble TGFβ3 by performing binding interactions with its cognate receptor TβRII. Our study delineates the fact that an effective combination of chaperones or optimum concentration of compatible osmolyte, can efficiently abrogate competing aggregation pathways and help attain the native conformation of a cysteine rich cytokine in a facile manner.

Effects of Osmolytes on Ligand Binding to Dihydropteroate Synthase from Bacillus anthracis.

Osmolyte interactions with ligands can affect their affinity for proteins and are dependent upon the cosolute and the functional groups of the ligand. Here, we explored ligand binding to Bacillus anthracis dihydropteroate synthase (BaDHPS) under osmotic stress conditions. Osmolyte effects were specific to the cosolute and ligand, suggesting interaction of the osmolytes with the free ligands in solution. The association rates of pterin pyrophosphate were mostly unaffected by the osmolytes, except for a 2-fold decrease in the presence of 1 M trehalose, while the dissociation rates decreased in most osmolyte solutions. The viscosity and dielectric constant of the solution did not correlate with the effects of the osmolytes. Experimental results were compared with predicted preferential interaction coefficients (Δμ23/RT) between the osmolytes and ligands. The Δμ23/RT were able to predict the experimental data for most of the osmolytes. Trehalose and proline effects did not correlate with the predicted values, indicating that these two osmolytes may affect binding in more complex ways than simple preferential interactions. Additionally, osmolytes weakly interacted with the sulfa drug sulfathiazole, which altered its affinity for BaDHPS, suggesting that these types of weak interactions can also impact drug binding. As osmolytes affect ligands binding to two different folate cycle enzymes (DHFRs and DHPS), we predicted how ligand binding to other folate cycle enzymes will be altered by the presence of osmolytes.

Influence of osmolytes and ionic liquids on the Bacteriorhodopsin structure in the absence and presence of oxidative stress: A combined experimental and computational study

Understanding the folding and stability of membrane proteins is of great importance in protein science. Recently, osmolytes and ionic liquids (ILs) are increasingly being used as drug delivery systems in the biopharmaceutical industry. However, the stability of membrane proteins in the presence of osmolytes and ILs is not yet fully understood. Besides, the effect of oxidative stress on membrane proteins with osmolytes or ILs has not been investigated. Therefore, we studied the influence of osmolytes and ILs as co-solvents on the stability of a model membrane protein (i.e., Bacteriorhodopsin in purple membrane of Halobacterium salinarum), using UV–Vis spectroscopy and molecular dynamics (MD) simulations. The MD simulations allowed us to determine the flexibility and solvent accessible surface area (SASA) of Bacteriorhodopsin protein in the presence and/or absence of co-solvents, as well as to carry out principal component analysis (PCA) to identify the most important movements in this protein. In addition, by means of UV–Vis spectroscopy we studied the effect of oxidative stress generated by cold atmospheric plasma on the stability of Bacteriorhodopsin in the presence and/or absence of co-solvents. This study is important for a better understanding of the stability of proteins in the presence of oxidative stress.

The interaction strength of an intrinsically disordered protein domain with its binding partner is little affected by very different cosolutes.

A common feature of intrinsically disordered proteins (IDPs) is a disorder-to-order transition upon binding to other proteins, which has been tied to multiple benefits, including accelerated association rates or binding with low affinity, yet high specificity. Given the balanced equilibrium concentrations of folded and unfolded state of an IDP we asked the question if changes in the chemical environment, such as the presence of osmolytes or crowding agents, have a strong influence on the interaction of an IDP. Here, we demonstrate the impact of cosolutes on the interaction of the intrinsically disordered transcription factor c-Myb and its binding partner, the kinase-inducible interaction domain (KIX) of the CREB-binding protein. Temperature jump relaxation kinetics and microscale thermophoresis were employed in order to quantify the rate constants and the binding affinity of the c-Myb/KIX complex, respectively, in the presence of various cosolutes. We find the binding free energy of the c-Myb/KIX complex only marginally modulated by cosolutes, whereas the enthalpy and entropy of the interaction are very sensitive to the respective solvent conditions. For different cosolutes we observe substantial changes in enthalpy, both favorable and unfavorable, which are going with entropy changes largely compensating the enthalpy effects in each case. These characteristics might reflect a potential mechanism by which c-Myb offsets changes in the physico-chemical environment to maintain a roughly unaltered binding affinity.

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.

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.

Osmolyte-Induced Folding and Stability of Proteins: Concepts and Characterization.

It is well-known that the typical protein’s three-dimensional structure is relatively unstable in harsh conditions. A practical approach to maintain the folded state and thus improve the stability and activity of proteins in unusual circumstances is to directly apply stabilizing substances such as osmolytes to the protein-containing solutions. Osmolytes as natural occurring organic molecules typically called “compatible” solutes, based on the concept that they do not perturb cellular components. However, urea and guanidine hydrochloride (GuHCl) as denaturing osmolytes destabilize many macromolecular structures and inhibit functions. Several studies have been so far performed to explain the actual interaction of an osmolyte with a protein. The present review is aimed to achieve a collective knowledge of the progress arise in the field of osmolyte-protein interactions. The following is also an overview of the main techniques to measure protein stability in the presence of osmolytes.

Bifidobacterium adolescentis is intrinsically resistant to antitubercular drugs

Multiple mutations in the β subunit of the RNA polymerase (rpoβ) of Mycobacterium tuberculosis (Mtb) are the primary cause of resistance to rifamycin (RIF). In the present study, bifidobacterial rpoβ sequences were analyzed to characterize the mutations that contribute to the development of intrinsic resistance to RIF, isoniazid, streptomycin and pyrazinamide. Sequence variations, which mapped to cassettes 1 and 2 of the rpoβ pocket, are also found in multidrug-resistant Mtb (MDR Mtb). Growth curves in the presence of osmolytes and different concentrations of RIF showed that the bacteria adapted rapidly by shortening the growth curve lag time. Insight into the adapted rpoβ DNA sequences revealed that B. adolescentis harbored mutations both in the RIF pocket and in regions outside the pocket. The minimum inhibitory concentrations (MICs) and mutant prevention concentrations (MPCs) indicated that B. longum, B. adolescentis and B. animalis are resistant to antitubercular drugs. 3D-homology modeling and binding interaction studies using computational docking suggested that mutants had reduced binding affinity towards RIF. RIF-exposed/resistant bacteria exhibited variant protein profiles along with morphological differences, such as elongated and branched cells, surface conversion from rough to smooth, and formation of a concentrating ring.

A generalized purification step for viral particles using mannitol flocculation.

Vaccine manufacturing has conventionally been performed by the developed world using traditional unit operations like filtration and chromatography. There is currently a shift in the manufacturing of vaccines to the less developed world, requiring unit operations that reduce costs, increase recovery, and are amenable to continuous manufacturing. This work demonstrates that mannitol can be used as a flocculant for an enveloped and nonenveloped virus and can purify the virus from protein contaminants after microfiltration. The recovery of the virus ranges from 58 to 96% depending on virus, the filter pore size, and the starting concentration of the virus. Protein removal of 80% was achieved for the small nonenveloped virus using a 0.1 µm filter because proteins were not flocculated with the virus and flowed through the filter. It is hypothesized that mannitol dehydrates the viral surface by controlling the water structure surrounding the virus. Without the ability to become compact, as occurs with proteins, the virus aggregates in the presence of osmolytes and proteins do not. Osmolyte flocculation is a scalable process using high flux microfilters. It has been applied to both an enveloped and nonenveloped virus, making this process friendly to a variety of vaccine and gene therapy products. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1027-1035, 2018.

Foliar Application of Glycine Betaine Alters Sugar Metabolism of Wheat Leaves Under Prolonged Field Drought Stress

The present investigation was carried out to examine the alterations in contents of various sugars in a set of 19 wheat genotypes under drought stress. In addition, foliar application of glycine betaine (GB, 100 mM) was done to explore participation of this osmolyte in drought response mechanism. Drought stress caused a significant increase in total soluble sugars (TSS) and starch levels, at both maximum tillering and anthesis stages. Activities of sucrose phosphate synthase (Suc-P-syn) and sucrose synthase (Suc syn) were also increased in drought stressed plants at both the stages. Exogenous application of GB resulted in higher TSS under drought stress; however reduced levels of starch were observed at anthesis in GB treated plants. GB application caused a decline in activity of Suc-P-syn and Suc syn at both stages. The alterations in sugar levels of GB sprayed plants suggested that regulation of sugar metabolism could undergo modifications (upstream or downstream) in presence of osmolytes like GB. Genotype-specific response to GB spray resulted in yield benefits in selected genotypes under drought stress.