Salt Potentiates Methylamine Counteraction System to Offset the Deleterious Effects of Urea on Protein Stability and Function

Cellular methylamines are osmolytes (low molecular weight organic compounds) believed to offset the urea’s harmful effects on the stability and function of proteins in mammalian kidney and marine invertebrates. Although urea and methylamines are found at 2:1 molar ratio in tissues, their opposing effects on protein structure and function have been questioned on several grounds including failure to counteraction or partial counteraction. Here we investigated the possible involvement of cellular salt, NaCl, in urea-methylamine counteraction on protein stability and function. We found that NaCl mediates methylamine counteracting system from no or partial counteraction to complete counteraction of urea’s effect on protein stability and function. These conclusions were drawn from the systematic thermodynamic stability and functional activity measurements of lysozyme and RNase-A. Our results revealed that salts might be involved in protein interaction with charged osmolytes and hence in the urea-methylamine counteraction.     

Testing the paradigm that the denaturing effect of urea on protein stability is offset by methylamines at the physiological concentration ratio of 2:1 (urea:methylamines)

The intra- and extracellular urea concentration in many organisms is sufficiently high to destabilize (inhibit) many proteins, yet organisms survive and function. The generally accepted explanation is the counteracting hypothesis, which holds that methylamines stabilize proteins and oppose the deleterious effect of urea. The two osmolytes are typically found at 2:1 concentration ratio (urea:methylamine) under physiological conditions. Does this mean that this ratio holds for all proteins in a cell? The present study tests the counteracting hypothesis by determining the effects of urea and methylamines (trimethylamine N-oxide and sarcosine), singly and in combination at a concentration ratio of 2:1 (urea:methylamine) on the thermal denaturation equilibrium, native state<-->denatured state of three different proteins (alpha-lactalbumin, lysozyme and Ribonuclease-A). We show here that the molar concentration of a methylamine required to offset the denaturing effect of urea at a given concentration is different for different proteins.     

Intracellular organic osmolytes: function and regulation

Cells of almost all organisms accumulate organic osmolytes when exposed to hyperosmolality, most often in the form of high salt or urea. In this review, we discuss 1) how the organic osmolytes protect; 2) the identity of osmolytes in Archaea, bacteria, yeast, plants, marine animals, and mammals; 3) the mechanisms by which they are accumulated; 4) sensors of osmolality; 5) the signaling pathways involved; and 6) mutual counteraction by urea and methylamines.     

Water Stress, Osmolytes and Proteins

Organic osmolytes are small solutes used by cells of numerouswater-stressed organisms and tissues to maintain cell volume.All known osmolytes are amino acids and derivatives, polyolsand sugars, methylamines, and urea; unlike salt ions, most are"compatible," i.e., do not perturb macromolecules. In addition,some stabilize macromolecules and are "counteracting" towardsperturbants, e.g., methylamines can stabilize proteins and ligandbinding against perturbations by urea in elasmobranchs and mammaliankidney, and (our latest findings) high hydrostatic pressurein deep-sea animals. Methylamines appear to coordinate watermolecules tightly, resulting in osmolyte exclusion from hydrationlayers of peptide backbones. This makes unfolded protein conformationsentropically unfavorable (work of Timasheff, Galinski, Bolenand coworkers). These properties have led to proposed uses inbiotechnology, agriculture and medicine, including improvedbiochemical methods, in vitro rescue of misfolded proteins incystic fibrosis and prion diseases (work of Welch and others),and plants engineered for drought and salt tolerance. Theseproperties also explain some but not all of the considerablevariation in osmolyte composition among species with differentmetabolisms and habitats, and among and within mammalian tissuesin development.     

A comparison of the counteracting effects of glycine betaine and TMAO on the activity of RNase A in aqueous urea solution

Trimethylamine-N-oxide (TMAO) and glycine betaine are counteracting osmolytes found in cellular systems under osmotic stress, often in association with high urea concentrations. TMAO is a characteristic component of cartilaginous fish and marine molluscs, while glycine betaine is more widely distributed, occurring in plants, bacteria and the mammalian kidney. As part of a project to explain and understand the action of these methylamines, the RNase A-catalysed degradation of polyuridylic acid in the presence of urea and various osmolytes (0-1.0 M) was studied using (31)P Nuclear Magnetic Resonance spectroscopy. The decrease in reaction rate induced by urea could be fully recovered with 1 molar equivalent of trimethylamine-N-oxide or 1.4 molar equivalents of glycine betaine. These results indicate that the modification of RNase A activity induced by urea is not associated with gross irreversible structural changes and that both glycine betaine and trimethylamine-N-oxide have kinetically detectable counteracting effects.     

Decreasing urea∶trimethylamine N-oxide ratios with depth in chondrichthyes: a physiological depth limit?

In marine osmoconformers, cells use organic osmolytes to maintain osmotic balance with seawater. High levels of urea are utilized in chondrichthyans (sharks, rays, skates, and chimaeras) for this purpose. Because of urea's perturbing nature, cells also accumulate counteracting methylamines, such as trimethylamine N-oxide (TMAO), at about a 2∶1 urea∶methylamine ratio, the most thermodynamically favorable mixture for protein stabilization, in shallow species. However, previous work on deep-sea teleosts (15 species) and chondrichthyans (three species) found an increase in muscle TMAO content and a decrease in urea content in chondrichthyans with depth. We hypothesized that TMAO counteracts protein destabilization resulting from hydrostatic pressure, as is demonstrated in vitro. Chondrichthyans are almost absent below 3,000 m, and we hypothesized that a limitation in urea excretion and/or TMAO retention might play a role. To test this, we measured the content of major organic osmolytes in white muscle of 13 chondrichthyan species caught with along-contour trawls at depths of 50-3,000 m; the deepest species caught was from 2,165 m. Urea and TMAO contents changed significantly with depth, with urea∶TMAO declining from 2.96 in the shallowest (50-90 m) groups to 0.67 in the deepest (1,911-2,165 m) groups. Urea content was 291-371 mmol/kg in the shallowest group and 170-189 mmol/kg in the deepest group, declining linearly with depth and showing no plateau. TMAO content was 85-168 mmol/kg in the shallowest group and 250-289 mmol/kg in the deepest groups. With data from a previous study for a skate at 2,850 m included, a second-order polynomial fit suggested a plateau at the greatest depths. When data for skates (Rajidae) were analyzed separately, a sigmoidal fit was suggested. Thus, the deepest chondrichthyans may be unable to accumulate sufficient TMAO to counteract pressure; however, deeper-living specimens are needed to fully test this hypothesis.     

Osmotic effectors in kidneys of xeric and mesic rodents: corticomedullary distributions and changes with water availability

Summary Urea, sodium, the methylamines glycine betaine and glycerophosphorylcholine (GPC), and the polyols sorbitol and myo-inositol are reported to be the major osmolytes in kidneys of laboratory mammals. These were measured (millimoles per kilogram wet weight) in kidney regions and urines of three species of wild rodents with different dehydration tolerances: the pocket mousePerognathus parvus (xeric), voleMicrotus montanus (mesic), and deer mousePeromyscus m. gambeli (intermediate). In animals kept without water for 4–6 days, sodium, urea, betaine and GPC+choline were found in gradients increasing from cortex to outer to inner medulla in all species, withPerognathus having the highest levels. Sorbitol was high in the inner medulla but low in the cortex and outer medulla; inositol was highest in the outer medulla. Totals of methylamines and methylamines plus polyols in the medulla showed high linear correlations (positive) with urea and with sodium values.Whole medullae were analyzed at several time points inMicrotus andPeromyscus subject to water diuresis followed by antidiuresis. In 102 h diuresis inMicrotus, all osmolytes decreased except inositol; however, only urea, sodium and sorbitol reached new steady states within 24 h. Urea returned to initial values in 18 h antidiuresis, while other osmolytes required up to 90 h. InPeromyscus, all osmolytes except the polyols declined in diuresis (max. 78 h test period). During antidiuresis, urea and GPC+choline rose to initial values in 18 h, with sodium and betaine requiring more time. In plots of both species combined, total methylamines+polyols correlated linearly (positive) with sodium, and GPC+choline with urea.Estimates of tissue concentrations suggest that total methylamines+polyols can account for intracellular osmotic balance in all species in antidiuresis and that sufficient concentrations of methylamines may be present to counteract perturbing effects of urea on proteins.Abbrevations GPC Glycero-3-phosphorylcholine - TCA trichloroacetic acid - M+P methylamines plus polyols     

Counteracting effects of urea and methylamines in function and structure of skeletal muscle myosin

Myosin is an asymmetric protein that comprises two globular heads (S1) and a double-stranded alpha-helical rod. We have investigated the effects of urea and the methylamines trimethylamine oxide (TMA-O) and glycine betaine (betaine) on activity and structure of skeletal muscle myosin. K(+) EDTA ATPase activity of myosin was almost completely inhibited by urea (2M); TMA-O stimulated myosin activity, whereas betaine had no effect. When combined with urea (0-2M), TMA-O or betaine (1 M) effectively protected the ATPase activity of myosin against inhibition. Intrinsic fluorescence measurements showed that in urea or TMA-O (0-2M), there were no shifts in the center of mass of the fluorescence spectrum of myosin, despite a decrease in fluorescence intensity. However, these osmolytes at concentrations above 2M produced a red shift in the emission spectrum. Betaine alone did not alter the center of mass at any concentration tested up to 5.2M. Thus, modifications in ATPase activity induced by low concentrations of solutes (<2M) are not directly correlated with the modifications in myosin structure detected by fluorescence. Both methylamines (>or=1M) were also able to protect myosin structure against urea-induced effects (2-8M). Protection was not observed for S1, supporting the hypothesis that these osmolytes have a biphasic effect on myosin: at lower concentrations there is an effect on the globular portion (S1), and at higher concentrations there is an effect on the coiled-coil (rod) portion of myosin.     

Parvalbumin characterization from the euryhaline stingray Dasyatis sabina

The Atlantic stingray, Dasyatis sabina found along the Gulf of Mexico and southeastern Atlantic coasts, is a euryhaline species of elasmobranch. This species is able to osmotically compensate for changing environmental salinity by altering plasma and intracellular solutes, including urea and counteracting methylamines (betaine and TMAO). Parvalbumin (PV) is an intracellular protein that facilitates muscle relaxation by sequestering calcium. Determining the effects that in situ concentrations of urea (146 mM), betaine (62 mM), and TMAO (11 mM) have on PV function in marine and freshwater adapted populations of D. sabina could provide insight into intracellular correlates of euryhaline tolerance for this species. PV from marine and freshwater populations of D. sabina was identified and purified by SDS-PAGE, western blot analysis, and full amino acid sequence analysis. Both populations exhibited two PV isoforms, PV I (approximately 12.18 kDa mw) and PV II (11.96 kDa mw). PV dissociation constants (K(D)) were determined in the presence and absence of physiological concentrations of urea, betaine, and TMAO by fluorescence spectroscopy using the fluorescent Ca(2+) indicator fluo-3 which competes with PV for Ca(2+). Functional studies revealed PV I showed no significant changes in calcium binding from in situ muscle conditions, except in the presence of betaine. In contrast, PV II's ability to bind calcium was increased relative to physiological conditions in the presence of each osmolyte independently. Thus, it appears that organic osmolytes have isoform specific effects on PV function.     

Trimethylamine oxide, betaine and other osmolytes in deep-sea animals: depth trends and effects on enzymes under hydrostatic pressure.

Most shallow teleosts have low organic osmolyte contents, e.g. 70 mmol/kg or less of trimethylamine oxide (TMAO). Our previous work showed that TMAO contents increase with depth in muscles of several Pacific families of teleost fishes, to about 180 mmol/kg wet wt at 2.9 km depth in grenadiers. We now report that abyssal grenadiers (Coryphaenoides armatus, Macrouridae) from the Atlantic at 4.8 km depth contain 261 mmol/kg wet wt in muscle tissue. This precisely fits a linear trend extrapolated from the earlier data. We also found that anemones show a trend of increasing contents of methylamines (TMAO, betaine) and scyllo-inositol with increasing depth. Previously we found that TMAO counteracts the inhibitory effects of hydrostatic pressure on a variety of proteins. We now report that TMAO and, to a lesser extent, betaine, are generally better stabilizers than other common osmolytes (myo-inositol, taurine and glycine), in terms of counteracting the effects of pressure on NADH Km of grenadier lactate dehydrogenase and ADP Km of anemone and rabbit pyruvate kinase.     

Naturally occurring organic osmolytes: from cell physiology to disease prevention

Osmolytes are naturally occurring organic compounds, which represent different chemical classes including amino acids, methylamines, and polyols. By accumulating high concentrations of osmolytes, organisms adapt to perturbations that can cause structural changes in their cellular proteins. Osmolytes shift equilibrium toward natively-folded conformations by raising the free energy of the unfolded state. As osmolytes predominantly affect the protein backbone, the balance between osmolyte-backbone interactions and amino acid side chain-solvent interactions determines protein folding. Abnormal cell volume regulation significantly contributes to the pathophysiology of several disorders, and cells respond to these changes by importing, exporting, or synthesizing osmolytes to maintain volume homeostasis. In recent years, it has become quite evident that cells regulate many biological processes such as protein folding, protein disaggregation, and protein-protein interactions via accumulation of specific osmolytes. Many genetic diseases are attributed to the problems associated with protein misfolding/aggregation, and it has been shown that certain osmolytes can protect these proteins from misfolding. Thus, osmolytes can be utilized as therapeutic targets for such diseases. In this review article, we discuss the role of naturally occurring osmolytes in protein stability, underlying mechanisms, and their potential use as therapeutic molecules.     

Osmolytes protect mitochondrial F(0)F(1)-ATPase complex against pressure inactivation

We have previously reported that carbohydrates and polyols protect different enzymes against thermal inactivation and deleterious effects promoted by guanidinium chloride and urea. Here, we show that these osmolytes (carbohydrates, polyols and methylamines) protect mitochondrial F(0)F(1)-ATPase against pressure inactivation. Pressure stability of mitochondrial F(0)F(1)-ATPase complex by osmolytes was studied using preparations of membrane-bound submitochondrial particles depleted or containing inhibitor protein (IP). Hydrostatic pressure in the range from 0.5 to 2.0 kbar causes inactivation of submitochondrial particles depleted of IP (AS particles). However, the osmolytes prevent pressure inactivation of the complex in a dose-dependent manner, remaining up to 80% of hydrolytic activity at the highest osmolyte concentration. Submitochondrial particles containing IP (MgATP-SMP) exhibit low ATPase activity and dissociation of IP increases the hydrolytic activity of the enzyme. MgATP-SMP subjected to pressure (2.2 kbar, for 1 h) and then preincubated at 42 degrees C to undergo activation did not have an increase in activity. However, particles pressurized in the presence of 1.5 M of sucrose or 3.0 M of glucose were protected and after preincubation at 42 degrees C, showed an activation very similarly to those kept at 1 bar. In accordance with the preferential hydration theory, we believe that osmolytes reduce to a minimum the surface of the macromolecule to be hydrated and oppose pressure-induced alterations of the native fold that are driven by hydration forces.     

Distinctive Solvation Patterns Make Renal Osmolytes Diverse

The kidney uses mixtures of five osmolytes to counter the stress induced by high urea and NaCl concentrations. The individual roles of most of the osmolytes are unclear, and three of the five have not yet been thermodynamically characterized. Here, we report partial molar volumes and activity coefficients of glycerophosphocholine (GPC), taurine, and myo-inositol. We derive their solvation behavior from the experimental data using Kirkwood-Buff theory. We also provide their solubility data, including solubility data for scyllo-inositol. It turns out that renal osmolytes fall into three distinct classes with respect to their solvation. Trimethyl-amines (GPC and glycine-betaine) are characterized by strong hard-sphere-like self-exclusion; urea, taurine, and myo-inositol have a tendency toward self-association; sorbitol and most other nonrenal osmolytes have a relatively constant, intermediate solvation that has components of both exclusion and association. The data presented here show that renal osmolytes are quite diverse with respect to their solvation patterns, and they can be further differentiated based on observations from experiments examining their effect on macromolecules. It is expected, based on the available surface groups, that each renal osmolyte has distinct effects on various classes of biomolecules. This likely allows the kidney to use specific combinations of osmolytes independently to fine-tune the chemical activities of several types of molecules.     

Effects of osmolytes on hexokinase kinetics combined with macromolecular crowding: test of the osmolyte compatibility hypothesis towards crowded systems

We investigated the effect of compatible and non-compatible osmolytes in combination with macromolecular crowding on the kinetics of yeast hexokinase. This was motivated by the fact that almost all studies concerning the osmolyte effects on enzyme activity have been performed in diluted buffer systems, which are far from the physiological conditions within cells, where the cytosol contains several hundred mg protein ml(-1). Four organic (glycerol, betaine, TMAO and urea) and one inorganic (NaCl) osmolyte were tested. It was concluded that the effect of compatible osmolytes (glycerol, betaine and TMAO) on V(max) and K(M) was practically equivalent in pure buffer and in 200-250 mg BSA ml(-1) supporting the view that these small organic osmolytes do minimal perturbance on enzyme function in physiological solutions. The effect of urea on enzyme kinetics was not independent of protein concentration, since the presence of 250 mg BSA ml(-1) partly compensated the perturbing effect of urea. Even though the organic osmolytes glycerol, betaine and TMAO are generally considered compatible with enzyme function, especially glycerol did have a significant effect on hexokinase kinetics, decreasing both k(cat), K(M) and k(cat)/K(M). The osmolytes decreased k(cat)/K(M) in the order: NaCl>Urea>TMAO/glycerol>betaine. For the organic osmolytes this order correlates with the degree of exclusion from protein-water interfaces. Thus, the stronger the exclusion the weaker the perturbing effects on k(cat)/K(M).     

Effects of organic solvents, methylamines, and urea on the affinity for Pi of the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-ATPase of sarcoplasmic reticulum can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. In a previous report (de Meis, L., Alves, E., and Martins, O.B. (1980) Biochemistry 19, 4252-4261), it was shown that organic solvent such as dimethyl sulfoxide and glycerol cause a decrease in the apparent Km for Pi. In this report it is shown that a similar effect is obtained with the methylamines glycine betaine and trimethylamine N-oxide. The apparent Km value for Pi in totally aqueous medium and in the presence of either 6.4 M glycerol, 1.4 M dimethyl sulfoxide, 0.4 M trimethylamine N-oxide, or 1 M glycine betaine were found to be respectively 2.85, 0.52, 0.52, 0.81, and 0.93 mM at pH 6.2 and greater than 10.0, 1.08, 2.53, 3.05, and 2.05 mM at pH 7.5. In contrast to the effect of methylamines, urea caused an increase in the apparent Km for Pi. When mixed in the appropriate concentration ratio, the effect of either organic solvent or methylamines is cancelled by urea.     

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.     

Catalytic and structural modifications of sarcoplasmic reticulum and plasma membrane (Ca2+ + Mg2+)ATPases induced by organic solutes that accumulate in living systems

Organic solutes such as urea, methylamines, polyols and amino acid can accumulate in the cytoplasm of cells to compensate for hyperosmotic conditions in the external medium. Whereas urea is considered to be typical of solutes that destabilize structure and function of proteins, methylamines, polyols and some amino acids appear to have the opposite effect, and can also compensate for the perturbing effects of urea. These effects have been extensively analyzed for a variety of proteins in terms of global changes in enzyme structure and acceleration or inhibition of overall reaction rates. Here the influence of these solutes on sarcoplasmic reticulum and plasma membrane (Ca²⁺ + Mg²⁺)ATPases is reviewed. The focus is on the changes induced by perturbing and stabilizing solutes at specific steps of the catalytic cycles of these enzymes, which can run forward (leading to ATP hydrolysis) and backward (leading to ATP synthesis). Structural changes promoted by osmolytes are correlated with functional changes, especially those that are related to energy coupling.This review is dedicated to Prof. Carlos Chagas Filho, founder of the Institute of Biophysics, on the occasion of its 50th anniversary.     

Naturally occurring osmolytes modulate the nanomechanical properties of polycystic kidney disease domains

Polycystin-1 (PC1) is a large membrane protein that is expressed along the renal tubule and exposed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their structure. However, it is known that the native conformation of proteins can be stabilized by protecting osmolytes that are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here, we used single-molecule force spectroscopy to study directly the effects of several naturally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable decrease in the mechanical stability of human PKD domains. We found that protecting osmolytes such as sorbitol and trimethylamine N-oxide can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally homologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally occurring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical properties and may lead to changes in the activation of the associated polycystin-2 channel or other intracellular events mediated by PC1.     

Osmolyte effect on the stability and folding of a hyperthermophilic protein

Proteins are known to be stabilized by naturally occurring osmolytes such as amino acids, sugars, and methylamines. Here, we examine the effect of trimethylamine-N-oxide (TMAO) on the conformational stability of ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII), which inherently possesses high conformational stability. Heat- and guanidine hydrochloride-induced unfolding experiments demonstrated that the conformational stability of Tk-RNase HII in the presence of 0.5M TMAO was higher than that in the absence of TMAO at all examined temperatures. TMAO affected the unfolding and refolding kinetics of Tk-RNase HII to a similar extent. These results indicate that proteins are universally stabilized by osmolytes, regardless of their robustness, and suggest a stabilization mechanism by osmolytes, caused by the unfavorable interaction of osmolytes with protein backbones in the denatured state. Our results also imply that the basic protein folding principle is not dependent on protein stability and evolution.