Class Of Osmolytes Research Articles

Characterizing osmolyte chemical class hierarchies and functional group requirements for thermal stabilization of proteins

Osmolytes are naturally occurring organic compounds that protect cellular proteins and other macromolecules against various forms of stress including temperature extremes. While biological studies have correlated the accumulation of certain classes of osmolytes with specific forms of stress, including thermal stress, it remains unclear whether or not these observations reflect an intrinsic chemical class hierarchy amongst the osmolytes with respect to effects on protein stability. In addition, very little is known in regards to the molecular elements of the osmolytes themselves that are essential for their functions. In this study, we use differential scanning fluorimetry to quantify the thermal stabilizing effects of members from each of the three main classes of protecting osmolytes on two model protein systems, C-reactive protein and tumor necrosis factor alpha. Our data reveals the absence of a strict chemical class hierarchy amongst the osmolytes with respect to protein thermal stabilization, and indicates differential responses of these proteins to certain osmolytes. In the second part of this investigation we dissected the molecular elements of amino acid osmolytes required for thermal stabilization of myoglobin and C-reactive protein. We show that the complete amino acid zwitterion is required for thermal stabilization of myoglobin, whereas removal of the osmolyte amino group does not diminish stabilizing effects on C-reactive protein. These disparate responses of proteins to osmolytes and other small molecules are consistent with previous observations that osmolyte effects on protein stability are protein-specific. Moreover, the data reported in this study support the view that osmolyte effects cannot be fully explained by considering only the solvent accessibility of the polypeptide backbone in the native and denatured states, and corroborate the need for more complex models that take into account the entire protein fabric.

Taurine Induces an Ordered but Functionally Inactive Conformation in Intrinsically Disordered Casein Proteins

Intrinsically disordered proteins (IDPs) are involved in various important biological processes, such as cell signalling, transcription, translation, cell division regulation etc. Many IDPs need to maintain their disordered conformation for proper function. Osmolytes, natural organic compounds responsible for maintaining osmoregulation, have been believed to regulate the functional activity of macromolecules including globular proteins and IDPs due to their ability of modulating the macromolecular structure, conformational stability, and functional integrity. In the present study, we have investigated the effect of all classes of osmolytes on two model IDPs, α- and β-casein. It was observed that osmolytes can serve either as folding inducers or folding evaders. Folding evaders, in general, do not induce IDP folding and therefore had no significant effect on structural and functional integrity of IDPs. On the other hand, osmolytes taurine and TMAO serve as folding inducers by promoting structural collapse of IDPs that eventually leads to altered structural and functional integrity of IDPs. This study sheds light on the osmolyte-induced regulation of IDPs and their possible role in various disease pathologies.

Unfoldness of the denatured state of proteins determines urea: Methylamine counteraction in terms of Gibbs free energy of stabilization.

In many tissues and organisms, large amount of urea gets accumulated to maintain osmotic balance. To evade the threatening impact of urea, living organisms accumulate methylamines, a class of osmolytes, in proportion of 2:1 (urea:methylamine). To understand underlying cause(s) for protein-specific counteraction behavior, thermodynamic stability (ΔGDo) of three disulfide free proteins (myoglobin, bovine cytochrome c and barstar) in the mixture of urea and methylamine has been estimated from guanidinium chloride-(GdmCl) driven denaturation curves. Using the experimentally measured values of ΔGDo obtained in the presence of individual methylamines and urea, we predicted the molar ratio of urea and a methylamine required for perfect compensation for each of the proteins. Interestingly, for all proteins studied, a similar ratio has been observed for perfect compensation. The predicted ratio for perfect compensation in terms of thermodynamic parameters was about 2:1 M ratio of urea to methylamine. Furthermore, a partial counteraction was observed in the myoglobin and barstar. However, for bovine cytochrome c, perfect compensation was observed in both GdmCl- and heat-driven denaturations. Our observations clearly suggest that the counteraction phenomenon depends on the extent of the unfolding of the denatured states of proteins.

A molecular interplay for osmolytes-induced phase behaviour of poly (vinyl methyl ether)

Naturally occurring osmolytes are small organic compounds that affect the functioning of the macromolecule and ever-present in the living systems. Trimethylamine N-oxide (TMAO), betaine and sarcosine are among the family of methylamines which are a class of osmolytes that may lead to the alteration in the transition behaviour of thermo-responsive polymers (TRPs). In this context, we have extended our work in the direction of exploring influence of various methylamines on the lower critical solution temperature (LCST) of poly (vinyl methyl ether) (PVME). Herein this study, we used various biophysical techniques to explore the impactful aspect of methylamines on the PVME polymer. The structural integrity of the additive is found to play a major role in altering the phase transition temperature of PVME. These additives are observed to have high binding affinity for water as compared to the polymer which result in the swollen to collapsed transition of PVME at the lower temperatures than the typical LCST of the polymer. One of the common aspects in all the three additives is their positive and negative charges separation that allows their effective binding with the water molecules. Although, each osmolytes decreases the LCST of the PVME, however, highest decrease is observed with sarcosine and lowest for TMAO. In case of sarcosine, less sterically hindered N atom may directs the possibility of its H-bonding ability towards water as compared to that for TMAO and betaine containing sterically hindered N atom.

Effects of osmolytes on stable UUCG tetraloops and their preference for a CG closing base pair

ABSTRACTOsmolytes have the potential to affect the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in the cell. To contribute to the understanding of the in vivo function of RNA we observed the effects of different classes of osmolytes on the UNCG tetraloop motif. UNCG tetraloops are the most common and stable of the RNA tetraloops and are nucleation sites for RNA folding. They also have a significant thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing UUCG loops was monitored using UV-Vis spectroscopy in the presence of osmolytes with different chemical properties. Interestingly, all of the osmolytes tested destabilized the hairpins, but all had little effect on the thermodynamic preference for a CG base pair, except for polyethylene glycol (PEG) 200. PEG 200 destabilized the loop with the CG closing base pair relative to the loop with a GC closing base pair. The destabilization was linear with increasing concentrations of PEG 200, and the slope of this relationship was not perturbed by changes in the hairpin stem outside of the closing pair. This result suggests that in the presence of PEG 200, the UUCG loop with a GC closing base pair may retain some preferential interactions with the cosolute that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes may influence how osmolytes tune the stability, and thus the function of a secondary structure motif in vivo.

Solute accumulation in the deep-sea bacterium Photobacterium profundum.

The identity and amounts of intracellular solutes in the deep-sea bacterium Photobacterium profundum strain SS9 were studied using nuclear magnetic resonance techniques. P. profundum strain SS9, a moderate piezophile which grows optimally at 20-30 MPa primarily accumulated glutamate and betaine, with lesser amounts of alanine, beta-hydroxybutyrate (beta-HB) and oligomers composed of the beta-HB units when grown at 0.1 MPa to early stationary phase. When grown at the optimal pressure, the cells preferentially increased intracellular concentrations of beta-HB and beta-HB oligomers, while the amino acid pools remained relatively constant. Since the organic solutes increased with increasing external NaCl in the medium, they are functioning as osmolytes. The beta-HB molecules represent a novel class of osmolytes, termed 'piezolytes,' whose cellular levels responded to hydrostatic pressure as well as osmotic pressure. Factors such as cell growth stage and temperature were also examined for their effect on the solute distribution in these cells.

Extension growth of the water mold Achlya: interplay of turgor and wall strength.

When hyphae of the water mold Achlya were subjected to osmotic stress, imposed with polyethylene glycol (PEG)-300 or sucrose, turgor pressure fell in proportion to the increase in external osmotic pressure. There was no evidence of turgor regulation, even over a period of days, yet the extension rate was unaffected until turgor was reduced to less than a third of the normal level of 0.6-0.8 MPa (6-8 bars). Measurements of the pressure at which the hyphae burst indicate that they respond to osmotic stress by softening their apical cell walls, sustaining extension growth despite reduced turgor pressure. The effect of osmolytes excluded by the wall was very different; superfusion of growing hyphae with PEG-6000 or dextran-6000 reduced turgor and stopped extension but did not induce wall softening. Furthermore, the hyphae did not resume growth during an hour or more of continuous exposure to these substances. Although the two classes of osmolytes have the same effect on turgor, they may induce different strains within the cell wall; this might then affect the capacity of the organism to detect the drop in turgor or to soften its cell wall. The interplay between turgor and wall strength supports the proposition that turgor supplies the driving force for extension and that production of the standard hyphal form requires a balance between hydrostatic pressure and a resistive cell wall.