AMER. ZOOL.

, 41:699709 (2001)

Water Stress, Osmolytes and Proteins1

PAUL H. YANCEY2
Whitman College, Biology Department, Walla Walla, Washington 99362

SYNOPSIS. Organic osmolytes are small solutes used by cells of numerous waterstressed organisms and tissues to maintain cell volume. All known osmolytes are amino acids and derivatives, polyols and sugars, methylamines, and urea; unlike salt ions, most are compatible, i.e., do not perturb macromolecules. In addition, some stabilize macromolecules and are counteracting towards perturbants, e.g., methylamines can stabilize proteins and ligand binding against perturbations by urea in elasmobranchs and mammalian kidney, and (our latest ndings) high hydrostatic pressure in deep-sea animals. Methylamines appear to coordinate water molecules tightly, resulting in osmolyte exclusion from hydration layers of peptide backbones. This makes unfolded protein conformations entropically unfavorable (work of Timasheff, Galinski, Bolen and coworkers). These properties have led to proposed uses in biotechnology, agriculture and medicine, including improved biochemical methods, in vitro rescue of misfolded proteins in cystic brosis and prion diseases (work of Welch and others), and plants engineered for drought and salt tolerance. These properties also explain some but not all of the considerable variation in osmolyte composition among species with different metabolisms and habitats, and among and within mammalian tissues in development.

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INTRODUCTION Modern biology has revealed a remarkable similarity at the molecular level underlying the enormous diversity of life. Most biomolecular research has focused on the macromolecules. But these operate in vivo in a medium dominated by micromoleculesmost notably water, electrolytes, metabolites, and osmolytes (regulators of cell volume) (Fig. 1)forming a milieu with an evolutionary history and properties that are less studied (Yancey et al., 1982). Gerstein and Levitt (1998) state: When scientists publish models of biological molecules in journals, they usually draw their models . . . against a plain, black background. We now known that the background in which these molecules exist wateris just as important as they are. Yet even they overlook the other signicant micromolecules of the cellular milieu, with osmolytes in particular having important interactions with water and macromolecules. I will focus on these properties within the
1 From the Symposium Osmoregulation: An Integral Approach presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 48 January 2000, at Atlanta, Georgia. 2 E-mail: yancey@whitman.edu

context of osmolyte primary function: adapting cells to water stress (see other papers in this symposium for osmolyte regulation). Because of recent reviews (Yancey, 1994; Gilles, 1997; Somero and Yancey, 1997), I will present primarily new ndings, which continue to expand with many exciting discoveries and actual or proposed applications. Considered here as a cellular disturbance due to water loss, water stress arises from evaporation into air or osmosis into concentrated aqueous surroundings. The latter may be due to saline habitats, physiological function (e.g., concentrated salt in mammalian kidneys), extracellular freezing, or disease (e.g., diabetes). Some cells survive such osmotic stress, but initially they may show disturbances, e.g., reduced growth rates of bacteria (Record et al., 1998), mammalian renal cells (Yancey and Burg, 1990) and embryos (Dawson and Baltz, 1997). For cells that successfully adapt, three generalizations have emerged. First, during short-term water loss cells often restore volume with inorganic ions as osmolytes (Land et al., 1998) while upregulating stress (heat-shock) proteins (Petronini et al., 1993; Sheikh-Hamad et al., 1994;

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FIG. 1. Examples of organic osmolytes in the major categories: uncharged sugars and polyols; free amino acids and derivatives; methylsulfonium (not shown) and methylammonium types, with fully substituted amino acids called betaines (N-trimethylglycine); and urea. For other examples and distributions, see Borowitzka (1985), Clark (1985), Rhodes and Hanson (1993), Yancey (1985, 1994), Storey (1997).

Smith et al., 1999), possibly indicating disturbances in protein structures (Fig. 2). Second, under long-term water stress, organic osmolytes replace ions for volume regulation, while stress proteins decline (Fig. 2). Such osmolyte use is widespread, from some Archaea to mammalian tissues such as kidney and brain. High levels of inorganic ions appear to be incompatible with long-term normal protein function, as perhaps are stress proteins, which may pro-

vide no protection against osmotic stress (Yancey and Walsh, 1994, Gagnon et al., 1999). Third, these solutes are limited to a few chemical types (Yancey et al., 1982) shown Figure 1. Only the evolutionarily ancient halophilic Archaea, restricted to marginal habitats such as brine ponds, appear to rely extensively on inorganic osmolytes at high concentrations. They osmoconform to the brine by accumulating K up to 7 M, mostly exclude environmental NaCl, and have proteins with many acidic amino acids (e.g., glutamate) that bind both water and K , and which do not allow proper conformation in the absence of high K (Lanyi, 1974). COMPATIBILITY PROPERTIES What selective forces have resulted in this conservation of or convergence on similar organic osmolytes (Fig. 1), with the (metabolically cheaper) inorganic ions usually disfavored? The compatibility hypothesis of Brown and Simpson (1972), later extended by Clark, Somero, Wyn Jones and others (Wyn Jones et al., 1977; Yancey et al., 1982), recognizes that inorganic ions (especially NaCl) at high levels disrupt protein function in vitro, but the major osmolytes usually do not, even at several molar for some. K salts sometimes show intermediate stimulation coinciding with typical cellular K levels, but perturb at higher concentrations. These effects are

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FIG. 2. Time course of changes in cell contents of stress protein HSP70 (detected by Western blots) and the osmolytes shown (detected by HPLC), in MadinDarby canine kidney cells in culture exposed to medium increased at time 0 from normal 290 mosm/kg to 500 mosm/kg with NaCl (* P 0.05 compared to previous time point) (Yancey, Walsh and Saari, unpublished).

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FIG. 3. Compatible and counteracting solute effects. A: Maximal activity of pig kidney argininosuccinase in vitro; modied from Yancey (1992) (Bet betaine; * P 0.05 compared to control). B: Osmoprotection by exogenous betaine measured as growth rate of E. coli relative to that in normal medium; modied from Record et al. (1998a).
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similar with enzymes from species without osmolytes, leading to an important corollary: effects of salt and organic osmolytes should be general features of protein-solutewater interactions, not of specic adaptations in proteins structure. Use of organic osmolytes should maintain functions without signicant disruptions over a wide range of external salinities. An example is shown Figure 3A; new studies continue to provide support. General compatibility has been tested in vivo by manipulating cell osmolytes, hypothesizing that cells under hyperosmotic stress will suffer if osmolyte levels are reduced, or will do better if given osmoprotectants (exogenous compatible osmolytes or precursors). The eubacterium E. coli, for which growth slows with increasing external osmolality, uses K and glutamate as major osmolytes. Salt inhibition of DNAprotein binding may be relieved in vivo by increased concentration of reactants in shrunken cells (Record et al., 1998b). Nevertheless, cell growth is inhibited, and is greatly improved by extracellular betaine, which through uptake replaces cellular K and glutamate (Fig. 3B) (Record et al., 1998a). Similarly, one line of mammalian renal medullary cells (PAP-HT25) in hyperos-

motic culture uses primarily sorbitol as an osmolyte. When production of sorbitol was inhibited, cell growth dropped in parallel with declining cell sorbitol content (inhibition had no effect in normal medium, in which these cells use little sorbitol) (Yancey et al., 1990a). However, addition of betaine (normally absent) to the medium largely reversed the decreased viability, in parallel to betaine uptake (Moriyama et al., 1991). Another line of renal cells suffered when deprived of myo-inositol, but improved when betaine was provided (Kitamura et al., 1997). With more normal cells of rat renal medulla, in both primary cultures (Rohr et al., 1999) and in vivo (Yancey et al., 1990b), inhibition of sorbitol synthesis triggered a compensating increase in betaine, such that there were no osmotic imbalances or apparent damage. Among non-renal cells, mouse hybridoma cells grew better in hyperosmotic media when given betaine, sarcosine, or dimethylglycine; protection increased with higher methylation (yaas et al., 1994a). Providing betaine to cultured SV-3T3 cells reduced stress protein expression and enhanced survival in osmotic shock (Petronini et al., 1993). Several studies have shown that mammalian embryos, exposed in vivo to hyperosmotic oviductal uids, grow bet-

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PAUL H. YANCEY Perturbing solutes Some organic osmolytes are able to offset some effects of solutes that perturb macromolecules. As organic osmolyte or waste product, urea (Fig. 1) is such a perturbant. At concentrations in marine cartilaginous shes and mammalian kidneys, urea alters many macromolecular structures and functions (usually disrupting). However, non-estivating urea-accumulating animals (cartilaginous shes, amphibia, mammalian kidneys) usually have other osmolytes, mainly methylamines such as trimethylamine Noxide (TMAO; Fig. 1). Our rst studies showed methylamines to exhibit not just simple compatibility, but often enhancement of protein activity and stability. This property was additive with urea effects such that they counteract, most effectively at about a 2:1 urea:TMAO ratio (similar to physiological levels, about 400: 200 mM in cartilaginous shes) (Yancey et al., 1982). These discoveries have been extensively conrmed in a variety of systems, some shown in Figures 3A and 4A, B. TMAO is usually a better stabilizer than other osmolytes including betaine (Yancey, 1994; Sackett, 1997; Goller and Galinski, 1999), perhaps explaining why TMAO is preferred in these shes. Like compatibility, counteraction occurs whether a protein is from a urea-accumulating tissue or not, and thus may reect universal mechanisms (Yancey et al., 1982). The counteracting-osmolytes hypothesis proposes that a mixture is more benecial than urea or methylamine alone, since the latter might overstabilize (Yancey et al., 1982). Using cultured renal cells, we found that adding high urea or betaine alone to the medium greatly reduced cell growth. However, adding both partly or fully restored normal growth (Yancey and Burg, 1990). In vivo, the mammalian renal medulla appears to regulate one of its methylamine osmolytes, glycerophosphorylcholine (GPC), to match urea contents (Peterson et al., 1992). Methylamines can sometimes offset perturbing salt effects. Pollard and Wyn Jones (1979) rst showed glycine and methylated derivatives counteracted NaCl inhibited of

ter in vitro in hyperosmotic media when provided organic osmolytes (Dawson and Baltz, 1997). STABILIZING AND COUNTERACTING PROPERTIES Many studies, often unrelated to osmolyte research, have shown the ability of these types of solutes to stabilize macromolecular structure in vitro in a variety of systems and disturbances (Yancey, 1994). While many studies use solutes well above physiological levels, in four natural stressesanhydrobiosis, extreme temperatures, perturbing solutes, and high hydrostatic pressurethere is good evidence that stabilizing properties (beyond simple compatibility) are important in vivo. Such solute effects are variously termed counteracting (Yancey et al., 1982), compensatory (Gilles, 1997), or chemical chaperoning (Brown et al., 1996). Anhydrobiosis Disaccharides, most notably trehalose, build up in anhydrobiotic dormant organisms (e.g., bakers yeast). However these sugars do not follow the mechanisms of non-interactive compatibility. Rather they bind to macromolecules and membranes, effectively replacing water. See Crowe et al., 1992, for details. Extreme temperature For freezing adaptations, see Storey (1997). Though not a water stress per se, high-temperature stress reveals key properties. Numerous studies show that most osmolytes increase protein thermal stability in vitro (reviewed by Yancey, 1994). Methylamines (Yancey, 1994) and trehalose (SolaPenna and Meyer-Fernandes, 1998) are often most effective. In vivo, carbohydrates may dominate: the hyperthermophilic archaeon Pyrococcus accumulates -mannosylglycerate with osmotic stress and dimyo-inositol phosphate with thermal stress; the latter solute is a potent protein thermostabilizer (Martin et al., 1999). And heat stress induces accumulation of sorbitol in at least one insect (Wolfe et al., 1998) and trehalose in yeast (De Virgilio et al., 1991).

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FIG. 4. Counteracting effects of methylamines in vitro (* P 0.05 compared to rabbit pyruvate kinase; urea used at 1 M in all tests (Ur urea, Bet betaine, GPC (modied from Burg et al., 1996). B: Polymerizaton of rat brain tubulin (modied Effects of 250 mM TMAO and high hydrostatic pressure on lactate dehydrogenase macrourid sh (modied from Gillett et al., 1997).

control). A: ADP Km of glycerophosphocholine) from Sackett, 1997). C: (LDH) from a deep-sea

a plant enzymes activity, with protection increasing with methylation. Many other studies show partial or full counteraction by methylamines of salt inhibition, including complex systems; e.g., TMAO (more than betaine) reverses the salt disruption of barnacle muscle ber architecture (Clark, 1985) and inhibited force in mammalian muscle bers (Nosek and Andres, 1998). High hydrostatic pressure The newest counteraction example is in the deep sea, where high hydrostatic pressure can perturb proteins (Siebenaller and Somero, 1989). In shallow marine animals, TMAO is usually found at 100 mmol kg 1 wet wt. (except in cartilaginous sh). Recently we found deep-sea teleosts and other animals have up to 300 mmol kg 1 (Fig. 5A), increasing with depth and thus perhaps related to pressure (Gillett et al., 1997; Kelly and Yancey, 1999). Testing found that, unlike glycine, TMAO offset pressure-inhibited stability of several LDH homologues (Yancey and Siebenaller, 1999) and polymerization of actin (Yancey et al., 2001). TMAO also offset pressure-induced increases in enzyme Km values (Fig. 4C). We recently found that some deep-sea species do not have TMAO, possibly because their taxa lack synthesis pathways. However they do have high levels of rare osmolytes, including scyllo-inositol in echinoderms, and hypotaurine and methyltaur-

ine in vestimentiferans (Yin et al., 1999). We are currently testing the effects of these on proteins under pressure. OSMOLYTE MECHANISMS A key premise of the compatible and counteracting hypotheses is they should involve universal water-solute-macromolecule interactions, the initial explanation proposed independently by Clark (see Clark, 1985) and Wyn Jones and co-workers (1977). They noted that amino acid and methylamine osmolytes are structurally similar to Hofmeister ions, which form a ranking of anions and cations based on consistent solution effects: kosmotropes [such as N(CH3)4 ] generally salt out, enhance catalysis by, and stabilize macromolecules, while chaotropes [such as Mg2 ] do the opposite (Collins and Washabaugh, 1985). Effects are additive, leading to counteraction if opposing ion types are paired. Of note is that both methylated organic osmolytes and inorganic ions are usually the best stabilizers. These universal properties are only partly understood. Inorganic and organic chaotropes may be attracted to protein functional groups; e.g., urea appears to unfold proteins by hydrogen bonding (Wu and Wang, 1999). Or they may bind to water less well than water does to itself, resulting in sequestering of solute away from bulk water (since water-water binding is more favor-

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FIG. 5. Patterns of osmolyte distributions. A: TMAO in muscles of deep-sea animals; open symbols are teleost shes (* P 0.05 compared to lower-depth value; modied from Kelly and Yancey, 1999). B: Changes in dominant osmolytes in rat heart and cerebrum during development; age 21 is weaning, age 77 is adult (modied from Miller et al., 2000 (* P 0.05 compared to value at previous time).

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able than water-solute binding) towards surfaces. This preferential interaction will unfold macromolecules because that maximizes favored surface interactions. In contrast, organic kosmotropes (except for carbohydrates in anhydrobiosis) exhibit an empirical tendency to be excluded from a proteins hydration shell (Timasheff, 1992). This preferential exclusion creates an entropically unfavorable order with

neighboring regions of high and low solute (Fig. 6). Proteins reduce this order by minimizing their exposed surface areas, by folding more compactly, by aggregation or precipitation (salting out); i.e., they will be stabilized. Binding of ligands to active sites will be favored if this involves loss of bound water. This would explain counteraction of pressure also (Yancey and Siebenaller, 1999), as pressure inhibits release of

FIG. 6. Model of solute and pressure effects on protein folding (based on models of Low, 1985; Timasheff, 1992; and Noto et al., 1995). Small dots represent water molecules. Addition of TMAO (t), excluded from the protein hydration layer, favors folding since that reduces the amount of excluded ordered water (middle). Addition of urea (u) enhances unfolding since it maximizes favorable binding sites (right). Differences in free energy between unfolded and native RNase T1 for the peptide backbone in solutions of 1 M urea, 0.5 M TMAO, or both (Net); showing that urea favors unfolding, TMAO favors folding and can offset the urea effect (data from Wang and Bolen, 1997). * Pressure can favor unfolding (left) but only if there is a net expansion as waters are released during folding.

OSMOLYTES hydration water from substrates and proteins in cases where volume expansion occurs (Fig. 6) (Siebenaller and Somero, 1989). The forces causing preferential exclusion are incompletely understood, with several proposed mechanisms in two broad categories (from Low, 1985; and Timasheff, 1992): (1) Steric exclusion: any large dissolved solute takes up more space than a water molecule, and if binding well to water and not attracted to a protein, it will be less able to pack next to it than water. This model may apply to carbohydrate osmolytes, whose -OH groups may t them into the water lattice readily, effectively replacing water molecules, but with a poorer geometrical t than water at protein surfaces. (2) Water structuring: Hofmeister (Collins and Washabaugh, 1985) and zwitterionic organic stabilizers may bind to water molecules better than to other solutes or than water to itself, and enhance water-water hydrogen bonding beyond their immediate hydration layer. Evidence includes reduced translational self-diffusion of water in osmolyte solutions (Clark, 1985), the tendency of most stabilizers to raise water surface tension (Timasheff, 1992), and molecular dynamic simulations showing that TMAO tightly coordinates water molecules (Noto et al., 1995). Methylamines often show the strongest effects; e.g., they elute faster than predicted from polyacrylamide gel columns, indicating rmly bound hydration shells (Galinski et al., 1997). Recently Wang and Bolen (1997) have shown that unfavorable interactions between TMAO and peptide backbones in particular explain the strong exclusion of TMAO, and thus its enhancement of protein folding (Fig. 6). Finally, Wiggins (1997) proposes that hydrophobic methyl groups can help alter water structure to favor native protein conformations only when on a charged atom, which repels these groups from each other to prevent hydrophobic aggregation. PRACTICAL APPLICATIONS Properties of osmolytes are increasingly useful in molecular biology and biotechnology. Stabilizing osmolytes improve re-

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constitution of functioning prokaryotic and eukaryotic protein-membrane complexes (Maloney and Ambudkar, 1989) and improve crystallization of proteins (Jeruzalmi and Steitz, 1997). Non-detergent sulfobetaines (similar to methylated taurines) are similarly useful with membrane proteins (Vuillard et al., 1998). Betaine improves cell-free transcription including PCR (Henke et al., 1997; Hethke et al., 1999), and in combination with hyperosmotic medium, it maximized monoclonal antibody production by hybridoma cells (yaas et al., 1994b). Finally, certain osmolytes such as TMAO work better than others in stabilizing proteins in freeze-thaw cycles (Goller and Galinski, 1999). Also, medically related aspects of osmolytes are increasingly important. Hyperglycemia in diabetes mellitus leads to buildup of the osmolyte sorbitol, with implications for treatment (Burg and Kador, 1988; Yancey et al., 1990 a, b). Pathogenic bacteria have been found to use trace amounts of human urinary osmolytes (including betaine, presumably from the kidney) to grow well in the presence of salts and urea. This could play a role in urinary tract infections and in drug design for treatment (Peddie et al., 1999). Welch and colleagues have suggested that osmolytes as chemical chaperones might rescue misfolded proteins in human diseases. They showed that 75100 mM TMAO can indeed restore function of one form of cystic brosis mutant protein (Fig. 7A) (Brown et al., 1996), and can inhibit the formation of aberrant protein aggregates in scrapie prion disease (Tatzelt et al., 1996). Conversely, we have found that GPC and TMAO can enhance formation of the undesirable -amyloid complex (Fig. 7B), associated with Alzheimers disease. Interestingly, the methylamines GPC, creatine and choline are excessively high in brains of Alzheimers patients (Wurtman, 1992; Pfefferbaum et al., 1999). A nal application is in agriculture. Transfer of genes for osmolyte production from salt-tolerant into salt-intolerant species is being used to adapt plants for saline and drought conditions (Tarczynski et al., 1993).

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FIG. 7. Protein-folding enhancements by osmolytes. A: Cl transport rate of the F508 mutant cystic brosis transmembrane-conductance regulator (CFTR) in vitro (modied from Brown et al., 1996) (* P 0.05 compared to wild-type control; P 0.05 compared to mutant control). B: Formation of amyloid brils in solution from -amyloid fragment 142, monitored as turbidity in two different runs (Yancey and Fyfe-Johnson, unpublished).

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EXCEPTIONS, OTHER FUNCTIONS, AND UNANSWERED QUESTIONS It seems likely that the use of organic osmolytes should speed up evolutionary adaptation relative to the alternative of altering most cell macromolecules to function in a dehydrated or concentrated ion solution (Yancey et al., 1982). However, there are some exceptions and inconsistencies involving compatibility and counteraction. For example, as universal as preferential exclusion may be for stabilizers, it does not always correlate with effects on protein function (Yancey, 1994; Randall et al., 1998; Burg et al., 1999). Wiggins (1997) proposes that gross protein stability may be less important than (poorly understood) solvation properties of active sites in explaining osmolyte effects on protein function at physiological concentrations. Remaining to be fully explained is why osmolytes in freezing adaptation are primarily carbohydrates (Storey, 1997), and why many organisms in other water stresses use mixtures of osmolytes, often of different classes. The need to cope with both high salt and urea probably explains why renal cells use both compatible polyols and counteracting methylamines (Bagnasco et al.,

1986). However, why do renal cells seem to regulate GPC but not betaine with urea (above)? Are betaine, sorbitol and inositol equivalent in compatibility (as suggested by renal experiments, above)? And while GPC does counteract urea, there is yet no consistent evidence that is a better stabilizer than betaine (Fig. 4A) (Burg et al., 1996). Finally, it is clear that some osmolytes have unique non-osmotic roles other than counteraction. Taurine, for example, high in mammalian heart and brain (Fig. 5B) and many marine invertebrates, may be an antioxidant (among other properties) (reviewed by Miller et al., 2000). Proline accumulation in water-stressed plants may be primarily for maintaining redox states than for compatibility or stabilizing aspects (see Cushman, 2001). And cyclitols that aid plants in water retention may also scavenge free radicals generated during drought and cold and other stresses (Orthen et al., 1994). Finally, there is evidence that TMAO can induce the formation of S-S bonds in proteins via hydrogen bonding () in the reaction SHON S H ON, creating reactive suldes (Brzezinksi and Zundel, 1993). But in many cases the selective rationales for certain osmolyte patterns and

OSMOLYTES types in certain organisms are not known. For example, for unclear reasons, contents of osmolyte-type compounds differ and change dramatically in different ways among mammalian tissues during development (Fig. 5B). Further studies on unique properties of osmolytes need to be conducted. REFERENCES
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