Calorimetric demonstration of the potential of molecular crowding to emulate the effect of an allosteric activator on pyruvate kinase kinetics

A method based on isothermal calorimetry is described for the direct kinetic assay of pyruvate kinase. In agreement with earlier findings based on the standard coupled assay system for this enzyme in the presence of a fixed ADP concentration, the essentially rectangular hyperbolic dependence of initial velocity upon phosphoenolpyruvate concentration is rendered sigmoidal by the allosteric inhibitor phenylalanine. This effect of phenylalanine can be countered by including a high concentration of a space-filling osmolyte such as proline in the reaction mixtures. This investigation thus affords a dramatic example that illustrates the need to consider potential consequences of thermodynamic nonideality on the kinetics of enzyme reactions in crowded molecular environments such as the cell cytoplasm.     

Mixed osmolytes: the degree to which one osmolyte affects the protein stabilizing ability of another

Mixtures of organic osmolytes occur in cells of many organisms, raising the question of whether their actions on protein stability are independent or synergistic. To investigate this question it is desirable to develop a system that permits evaluation of the effect of one osmolyte on the efficacy of another to either force-fold or denature a protein. A means of evaluating the efficacy of an osmolyte is provided by its m-value, an experimental quantity that measures the ability of the osmolyte to force a protein to unfold or fold. An experimental system is presented that enables evaluations of the m-values of osmolytes in the presence and absence of a second osmolyte. The experimental system involves use of a marginally stable protein in 10 mM buffer (pH 7, 200 mM salt, and 34 degrees C) that is at the midpoint of its native to denatured transition. These conditions enable determination of m-values for protecting and denaturing osmolytes in the presence and absence of a second osmolyte, permitting assessment of the extent to which the two osmolytes affect each other's efficacy. The two osmolytes investigated in this work are the denaturing osmolyte, urea, and the protecting osmolyte, sarcosine. Results show unequivocally that neither osmolyte alters the efficacy of the other in forcing the protein to fold or unfold-the osmolytes act independently on the protein despite their combined concentrations being in the multi-molar range. These osmolytes avoid altering one another's efficacy at these high concentrations because the number of osmolyte interaction sites on the protein is large and the binding constants are quite small. Consequently, the site occupancies are low enough in number that the two osmolytes neither compete nor cooperate in interacting with the protein.     

The existence of molecular water pumps in the nervous system: a review of the evidence

Recently, the presence if both influx and efflux molecular water pumps (MWP's) in vertebrate cells has been reported. These appear to use a common mechanism; the intercompartmental cotransport of water uphill against a gradient as a hydrophylic osmolyte is transported down its own gradient, in a regulated fashion, by a membrane spanning cotransporter protein. In each case, the dwell time of the transported osmolyte is short in that it is metabolically converted and its products either eliminated or recycled, thereby maintaining the required high intercompartmental gradient. An influx water pump osmolyte has been identified as a sodium-glucose complex, and an efflux water pump osmolyte as N-acetylhistidine. These osmolytes may also be archetypal representatives of many other osmolytes with similar functions in a variety of cells. When recycled, the osmolyte metabolites appear to be dewatered during high affinity binding that is associated with their active transport back across the membrane prior to intracellular resynthesis of the osmolyte. Since these cyclical systems result in the pumping of water, they also appear to create a previously unrecognized motive force which results in the establishment of unidirectional transcellular water flows between apical and basolateral cell membranes. As neurons represent highly specialized forms of animal cells, and cells which are also extremely sensitive to changes in osmotic pressure, the presence of these water pumps in the CNS could be significant. There would be connotations with regard to how neurons regulate water balance and transaxonal flow as well as to how these factors affect the integrated function of the nervous system. In this article, evidence of the presence of MWP's in the nervous system, and how they might relate to aspects of both normal and abnormal brain function is reviewed.     

An osmolyte mitigates the destabilizing effect of protein crowding

Most theories predict that macromolecular crowding stabilizes globular proteins, but recent studies show that weak attractive interactions can result in crowding-induced destabilization. Osmolytes are ubiquitous in biology and help protect cells against stress. Given that dehydration stress adds to the crowded nature of the cytoplasm, we speculated that cells might use osmolytes to overcome the destabilization caused by the increased weak interactions that accompany desiccation. We used NMR-detected amide proton exchange experiments to measure the stability of the test protein chymotrypsin inhibitor 2 under physiologically relevant crowded conditions in the presence and absence of the osmolyte glycine betaine. The osmolyte overcame the destabilizing effect of the cytosol. This result provides a physiologically relevant explanation for the accumulation of osmolytes by dehydration-stressed cells.     

An effective solvent theory connecting the underlying mechanisms of osmolytes and denaturants for protein stability

An all-atom Gō model of Trp-cage protein is simulated using discontinuous molecular dynamics in an explicit minimal solvent, using a single, contact-based interaction energy between protein and solvent particles. An effective denaturant or osmolyte solution can be constructed by making the interaction energy attractive or repulsive. A statistical mechanical equivalence is demonstrated between this effective solvent model and models in which proteins are immersed in solutions consisting of water and osmolytes or denaturants. Analysis of these studies yields the following conclusions: 1), Osmolytes impart extra stability to the protein by reducing the entropy of the unfolded state. 2), Unfolded states in the presence of osmolyte are more collapsed than in water. 3), The folding transition in osmolyte solutions tends to be less cooperative than in water, as determined by the ratio of van 't Hoff to calorimetric enthalpy changes. The decrease in cooperativity arises from an increase in native structure in the unfolded state, and thus a lower thermodynamic barrier at the transition midpoint. 4), Weak denaturants were observed to destabilize small proteins not by lowering the unfolded enthalpy, but primarily by swelling the unfolded state and raising its entropy. However, adding a strong denaturant destabilizes proteins enthalpically. 5), The folding transition in denaturant-containing solutions is more cooperative than in water. 6), Transfer to a concentrated osmolyte solution with purely hard-sphere steric repulsion significantly stabilizes the protein, due to excluded volume interactions not present in the canonical Tanford transfer model. 7), Although a solution with hard-sphere interactions adds a solvation barrier to native contacts, the folding is nevertheless less cooperative for reasons 1–3 above, because a hard-sphere solvent acts as a protecting osmolyte.     

Osmolyte trimethylamine-N-oxide does not affect the strength of hydrophobic interactions: origin of osmolyte compatibility

Osmolytes are small organic solutes accumulated at high concentrations by cells/tissues in response to osmotic stress. Osmolytes increase thermodynamic stability of folded proteins and provide protection against denaturing stresses. The mechanism of osmolyte compatibility and osmolyte-induced stability has, therefore, attracted considerable attention in recent years. However, to our knowledge, no quantitative study of osmolyte effects on the strength of hydrophobic interactions has been reported. Here, we present a detailed molecular dynamics simulation study of the effect of the osmolyte trimethylamine-N-oxide (TMAO) on hydrophobic phenomena at molecular and nanoscopic length scales. Specifically, we investigate the effects of TMAO on the thermodynamics of hydrophobic hydration and interactions of small solutes as well as on the folding-unfolding conformational equilibrium of a hydrophobic polymer in water. The major conclusion of our study is that TMAO has almost no effect either on the thermodynamics of hydration of small nonpolar solutes or on the hydrophobic interactions at the pair and many-body level. We propose that this neutrality of TMAO toward hydrophobic interactions-one of the primary driving forces in protein folding-is at least partially responsible for making TMAO a "compatible" osmolyte. That is, TMAO can be tolerated at high concentrations in organisms without affecting nonspecific hydrophobic effects. Our study implies that protein stabilization by TMAO occurs through other mechanisms, such as unfavorable water-mediated interaction of TMAO with the protein backbone, as suggested by recent experimental studies. We complement the above calculations with analysis of TMAO hydration and changes in water structure in the presence of TMAO molecules. TMAO is an amphiphilic molecule containing both hydrophobic and hydrophilic parts. The precise balance of the effects of hydrophobic and hydrophilic segments of the molecule appears to explain the virtual noneffect of TMAO on the strength of hydrophobic interactions.     

Subcellular compartmentation of proline in the leaves of the subantarctic Kerguelen cabbage Pringlea antiscorbutica R. Br. In vivo13C-NMR study

Proline is one of the major solutes accumulated upon salt stress in leaves, stem and roots of the subantarctic Brassicaceae Pringlea antiscorbutica R. Br. (Kerguelen cabbage). Using in vivo ¹³C-NMR techniques, it was possible for the first time to visualize the subcellular compartmentation of proline between cytoplasmic and vacuolar compartments in Pringlea leaves. We observed that this osmolyte accumulated at a 2–3 times higher concentration in the cytoplasm than in the vacuole.     

Osmolyte‐induced conformational changes in the Hsp90 molecular chaperone

Osmolytes are small molecules that play a central role in cellular homeostasis and the stress response by maintaining protein thermodynamic stability at controlled levels. The underlying physical chemistry that describes how different osmolytes impact folding free energy is well understood, however little is known about their influence on other crucial aspects of protein behavior, such as native‐state conformational changes. Here we investigate this issue with the Hsp90 molecular chaperone, a large dimeric protein that populates a complex conformational equilibrium. Using small angle X‐ray scattering we observe dramatic osmolyte‐dependent structural changes within the native ensemble. The degree to which different osmolytes affect the Hsp90 conformation strongly correlates with thermodynamic metrics of their influence on stability. This observation suggests that the well‐established osmolyte principles that govern stability also apply to large‐scale conformational changes, a proposition that is corroborated by structure‐based fitting of the scattering data, surface area comparisons and m‐value analysis. This approach shows how osmolytes affect a highly cooperative open/closed structural transition between two conformations that differ by a domain‐domain interaction. Hsp90 adopts an additional ligand‐specific conformation in the presence of ATP and we find that osmolytes do not significantly affect this conformational change. Together, these results extend the scope of osmolytes by suggesting that they can maintain protein conformational heterogeneity at controlled levels using similar underlying principles that allow them to maintain protein stability; however the relative impact of osmolytes on different structural states can vary significantly.     

Mutational and structural-based analyses of the osmolyte effect on protein stability

It is known that several naturally occurring substances known as osmolytes increase the conformational stability of proteins. Bolen and co-worker proposed the osmophobic theory, which asserts the osmolyte effect occurs because of an unfavorable interaction of osmolytes mainly with the protein backbone, based on the results on the transfer Gibbs energy of amino acids (Deltag) [Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963]. In this paper, we report the effect of sarcosine on the conformational stability (DeltaG) of RNase Sa (96 residues and one disulfide bond) and four mutant proteins. The thermal denaturation curves for RNase Sa in sarcosine fitted a two-state model on nonlinear least-squares analysis. All the RNase Sa proteins were stabilized by sarcosine. For example, the increase in stability of the wild-type protein in 4 M sarcosine due to the osmolyte effect (Delta(o)DeltaG) is 3.2 kcal/mol. Mutational analysis of the osmolyte effect indicated that the changed Delta(o)DeltaG values upon mutation (Delta(m)Delta(o)DeltaG), as estimated from the Deltag values, are similar to the experimental values. Structural-based analysis of the osmolyte effect was also performed using model denatured structures: (a) a fully extended model (single chain) with no disulfide bond, (b) two-part, unfolded models (two chains) with a disulfide bond constructed through molecular dynamic (MD) simulation, and (c) a two-part, folded model (two chains). The two-part, unfolded models were expected to be more suitable as denatured structures. The Delta(o)DeltaG values calculated using the two-part, unfolded models were more consistent with experimental values than those calculated using the fully extended and two-part, folded models. This suggests that MD simulation is useful for testing denatured structures. These results indicate that the osmophobic theory can explain the osmolyte effect on protein stability.     

Organization of early frog embryos by chemical waves emanating from centrosomes

The large cells in early vertebrate development face an extreme physical challenge in organizing their cytoplasm. For example, amphibian embryos have to divide cytoplasm that spans hundreds of micrometres every 30 min according to a precise geometry, a remarkable accomplishment given the extreme difference between molecular and cellular scales in this system. How do the biochemical reactions occurring at the molecular scale lead to this emergent behaviour of the cell as a whole? Based on recent findings, we propose that the centrosome plays a crucial role by initiating two autocatalytic reactions that travel across the large cytoplasm as chemical waves. Waves of mitotic entry and exit propagate out from centrosomes using the Cdk1 oscillator to coordinate the timing of cell division. Waves of microtubule-stimulated microtubule nucleation propagate out to assemble large asters that position spindles for the following mitosis and establish cleavage plane geometry. By initiating these chemical waves, the centrosome rapidly organizes the large cytoplasm during the short embryonic cell cycle, which would be impossible using more conventional mechanisms such as diffusion or nucleation by structural templating. Large embryo cells provide valuable insights to how cells control chemical waves, which may be a general principle for cytoplasmic organization.     

Osmotic regulation of Rab-mediated organelle docking

Osmotic gradients across organelle and plasma membranes modulate the rates of membrane fission and fusion; sufficiently large gradients can cause membrane rupture [1-6]. Hypotonic gradients applied to living yeast cells trigger prompt (within seconds) swelling and fusion of Saccharomyces cerevisiae vacuoles, whereas hypertonic gradients cause vacuoles to fragment on a slower time scale [7-11]. Here, we analyze the influence of osmotic strength on homotypic fusion of isolated yeast vacuoles. Consistent with previously reported in vivo results, we find that decreases in osmolyte concentration increase the rate and extent of vacuole fusion in vitro, whereas increases in osmolyte concentration prevent fusion. Unexpectedly, our results reveal that osmolytes regulate fusion by inhibiting early Rab-dependent docking or predocking events, not late events. Our experiments reveal an organelle-autonomous pathway that may control organelle surface-to-volume ratio, size, and copy number: Decreasing the osmolyte concentration in the cytoplasmic compartment accelerates Rab-mediated docking and fusion. By altering the relationship between the organelle surface and its enclosed volume, fusion in turn reduces the risk of membrane rupture.     

Beta-alanine but not taurine can function as an organic osmolyte in preimplantation mouse embryos cultured from fertilized eggs

Early preimplantation mouse embryos are susceptible to the detrimental effects of increased osmolarity and, paradoxically, their in vitro development is significantly compromised by osmolarities near that of oviductal fluid. In vitro development can be restored, however, by several compounds that are accumulated by 1-cell embryos to act as organic osmolytes, providing intracellular osmotic support and cell volume regulation. Taurine, a substrate of the beta-amino acid transporter that functions as an organic osmolyte transporter in other cells, had been proposed to function as an organic osmolyte in mouse embryos. Here, however, we found that taurine is neither able to provide protection for in vitro embryo development against increased osmolarity nor is it accumulated to higher intracellular levels as osmolarity is increased, indicating that it cannot function as an organic osmolyte in early preimplantation embryos. In contrast, beta-alanine, the other major substrate of the beta-amino acid transporter, both protects against increased osmolarity and is accumulated to somewhat higher levels as osmolarity is increased, indicating that it is able to function as an organic osmolyte in embryos. However, we also found that beta-alanine is displaced from embryos by glycine-the most effective organic osmolyte in embryos previously identified-and beta-alanine does not increase protection above that afforded by glycine at concentrations near those in vivo. Thus, the beta-amino acid transporter is likely present in early preimplantation embryos to supply beta-amino acids such as taurine for purposes other than to serve as organic osmolytes.     

Betaine is a highly effective organic osmolyte but does not appear to be transported by established organic osmolyte transporters in mouse embryos

Betaine protects early preimplantation mouse embryos against increased osmolarity in vitro, functioning as an organic osmolyte. Betaine is effective at very low external concentrations, with half-maximal protection of 1-cell embryo development to blastocysts at approximately 50 microM, making it one of the best osmoprotectants for mouse preimplantation embryos. We performed studies designed to determine whether known high-affinity organic osmolyte transporters could account for the ability of betaine to act as an organic osmolyte in preimplantation embryos. We found no evidence in 1-cell embryos of transport by a betaine/GABA transporter (BGT1), the osmoregulated betaine transporter found in a number of cell types, as betaine and GABA did not inhibit each other's transport. Instead, all saturable GABA transport in embryos was apparently via the beta-amino acid transporter. We also found that the glycine transporter, GLY, which mediates osmoprotective transport of glycine in early preimplantation embryos, does not appear to transport betaine. Finally, increased osmolarity did not induce any detectable System A amino acid transporter activity, which is osmotically-inducible in other cells and can transport betaine. There does appear, however, to be a saturable betaine transporter in 1-cell mouse embryos, as considerable 14C-betaine transport was measured which was substantially inhibited by excess unlabeled betaine. Our data imply that betaine functions as an organic osmolyte in embryos due to its saturable transport via a mechanism distinct from known osmolyte transporters. We propose that an unidentified high-affinity betaine transporter may be expressed in early embryos and mediate transport of betaine as an organic osmolyte.     

Protein folding and stability in the presence of osmolytes

Osmolytes are molecules whose function, among others, is to balance the hydrostatic pressure between the intracellular and extracellular compartments. Accumulation of osmolytes in a cell occurs in response to stress caused by changes in pressure, temperature, pH, or the concentration of inorganic salts. Osmolytes can prevent the denaturation of native proteins and promote the renaturation of unfolded proteins. Investigation of the roles of osmolyte in these processes is essential for our understanding of the mechanisms of protein folding and function in vivo. The large number of published reports that have been devoted to the effects of osmolytes on proteins are not always consistent with each other. In this review, an attempt is made to systemize the array of data on this subject and to consider the problem of protein folding and stability in osmolyte solutions from a single viewpoint.     

Ultraviolet B radiation induces cell shrinkage and increases osmolyte transporter mRNA expression and osmolyte uptake in HaCaT keratinocytes

We have previously shown that compatible organic osmolytes, such as betaine, myo-inositol and taurine, are part of the stress response of normal human keratinocytes (NHKs) to ultraviolet B (UVB) radiation. In this regard, we tested human HaCaT keratinocytes as a surrogate cell line for NHK. HaCaT cells osmo-dependently express mRNA specific for transport proteins for betaine (BGT-1), myo-inositol (SMIT) and taurine (TAUT). Compared to normoosmotic (305 mosmol/l) controls, which strongly constitutively expressed BGT-1 mRNA, strong induction of SMIT and TAUT mRNA as well as low induction of BGT-1 mRNA expression was observed between 3 and 9 h after hyperosmotic exposure (405 mosmol/l). This expression correlated with an increased osmolyte uptake. Conversely, hypoosmotic (205 mosmol/l) stimulation led to a significant efflux of osmolytes. Exposure to UVB (290-315 nm) radiation induced cell shrinkage which was followed by an upregulation of osmolyte transporter mRNA levels and osmolyte uptake. These results demonstrate that human HaCaT keratinocytes possess an osmolyte strategy including UVB-induced cell shrinkage and following increased osmolyte uptake. However, several differences in osmolyte transporter expression and uptake were noted between NHK and HaCaT cells, indicating that the use of HaCaT cells as a surrogate cell line for NHK has limitations.     

Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress.

Polyols, or polyhydroxy alcohols, are produced by many fungi. Saccharomyces cerevisiae produces large amounts of glycerol, and several fungi that cause serious human infections produce D-arabinitol and mannitol. Glycerol functions as an intracellular osmolyte in S. cerevisiae, but the functions of D-arabinitol and mannitol in pathogenic fungi are not yet known. To investigate the functions of mannitol, we constructed a new mannitol biosynthetic pathway in S. cerevisiae. S. cerevisiae transformed with multicopy plasmids encoding the mannitol-1-phosphate dehydrogenase of Escherichia coli produced mannitol, whereas S. cerevisiae transformed with control plasmids did not. Although mannitol production had no obvious phenotypic effects in wild-type S. cerevisiae, it restored the ability of a glycerol-defective, osmosensitive osg1-1 mutant to grow in the presence of high NaCl concentrations. Moreover, osg1-1 mutants producing mannitol were more resistant to killing by oxidants produced by a cell-free H2O2-FeSO4-NaI system than were controls. These results indicate that mannitol can (i) function as an intracellular osmolyte in S. cerevisiae, (ii) substitute for glycerol as the principal intracellular osmolyte in S. cerevisiae, and (iii) protect S. cerevisiae from oxidative damage by scavenging toxic oxygen intermediates.     

Characterization of volume-sensitive organic osmolyte efflux and anion current in Xenopus oocytes

Cell swelling activates an outwardly rectifying anion current in numerous mammalian cell types. An extensive body of evidence indicates that the channel responsible for this current is the major pathway for volume regulatory organic osmolyte loss. Cell swelling also activates an outwardly rectifying anion current in Xenopus oocytes. Unlike mammalian cells, oocytes allow the direct study of both swelling-activated anion current and organic osmolyte efflux under nearly identical experimental conditions. We therefore exploited the unique properties of oocytes in order to examine further the relationship between anion channel activity and swelling-activated organic osmolyte transport. Swelling-activated anion current and organic osmolyte efflux were studied in parallel in batches of oocytes obtained from single frogs. The magnitude of swelling-activated anion current and organic osmolyte efflux exhibited a positive linear correlation. In addition, the two processes had similar pharmacological characteristics and activation, rundown and reactivation kinetics. The present study provides further strong support for the concept that the channel responsible for swelling-activated Cl⁻ efflux and the outwardly rectifying anion conductance is also the major pathway by which organic osmolytes are lost from vertebrate cells during regulatory volume decrease. Received: 22 April 1996/Revised: 18 December 1996     

Temperature-mediated switching of protectant-denaturant behavior of trimethylamine-N-oxide and consequences on protein stability from a replica exchange molecular dynamics simulation study

The detailed mechanism of protein folding–unfolding processes with the aid of osmolytes has been a leading topic of discussion over many decades. We have used replica-exchange molecular dynamics simulation to propose the molecular mechanism of interaction of a 20-residue mini-protein with urea and trimethylamine N-oxide (TMAO) that act as denaturing and protecting osmolyte, respectively, in binary osmolyte solutions. Urea is found to exert its action by interacting directly with the protein residues. Temperature tolerance of TMAO’s action is particularly emphasised in this study. At lower range of temperature, TMAO acts as a successful protein protectant. Interestingly, the study discloses the tendency of TMAO molecules to prefer self-association at the protein surface at elevated temperature. A greater number of TMAO molecules in the protein hydration shell at higher temperature is also observed. Dihedral angle principal component analysis and free energy landscape plots sampled all possible conformations adopted by the protein that reveal highly folded behaviour of the protein in pure water and binary TMAO solutions and highly unfolded behaviour in presence of urea.     

Dissecting the roles of osmolyte accumulation during stress

Many plants accumulate organic osmolytes in response to the imposition of environmental stresses that cause cellular dehydration. Although an adaptive role for these compounds in mediating osmotic adjustment and protecting subcellular structure has become a central dogma in stress physiology, the evidence in favour of this hypothesis is largely correlative. Transgenic plants engineered to accumulate proline, mannitol, fructans, trehalose, glycine betaine or ononitol exhibit marginal improvements in salt and/or drought tolerance. While these studies do not dismiss causative relationships between osmolyte levels and stress tolerance, the absolute osmolyte concentrations in these plants are unlikely to mediate osmotic adjustment. Metabolic benefits of osmolyte accumulation may augment the classically accepted roles of these compounds. In re-assessing the functional significance of compatible solute accumulation, it is suggested that proline and glycine betaine synthesis may buffer cellular redox potential. Disturbances in hexose sensing in transgenic plants engineered to produce trehalose, fructans or mannitol may be an important contributory factor to the stress-tolerant phenotypes observed. Associated effects on photoassimilate allocation between root and shoot tissues may also be involved. Whether or not osmolyte transport between subcellular compartments or different organs represents a bottleneck that limits stress tolerance at the whole-plant level is presently unclear. None the less, if osmolyte metabolism impinges on hexose or redox signalling, then it may be important in long-range signal transmission throughout the plant.