Protein and DNA destabilization by osmolytes: The other side of the coin

Life Sciences 88 (2011) 117–125

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Protein and DNA destabilization by osmolytes: The other side of the coin

Laishram R. Singh a, Nitesh Kumar Poddar a, Tanveer Ali Dar b, Raj Kumar c, Faizan Ahmad a,⁎a Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi-110025, Indiab School of Biosciences and Biotechnology, Baba Ghulam Shah Badshah University, Rajouri, Jammu and Kashmir-185131, Indiac Department of Basic Sciences, The Commonwealth Medical College, Scranton, PA-18510, USA

⁎ Corresponding author. Tel.: +91 11 2698 1733; faxE-mail address: [email protected] (F. Ahma

0024-3205/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.lfs.2010.10.020

a b s t r a c t

Article history:Received 19 June 2010Accepted 19 October 2010Available online 1 November 2010

Keywords:OsmolytePreferential hydrationProtein stabilizationProtein destabilizationProtein folding

Osmolytes are naturally occurring small molecules accumulated intracellularly to protect organisms fromvarious denaturing stresses. Similar to the two faces of a coin, several of these osmolytes are stabilizing anddestabilizing proteins depending on the concentrations and/or solvent conditions. For example, the wellknown stabilizing osmolyte, trehalose destabilizes some proteins at high concentration and/or high pH. Inspite of the fact that destabilizing aspects of osmolytes can modulate many cellular processes includingregulation of protein homeostasis (proteostasis), protein–protein interaction, and protein–DNA interaction,researchers have mostly focused on the stabilizing aspects of osmolytes. Thus, it is important to look into bothaspects of osmolytes to determine their precise role under physiological conditions. In this article, we havediscussed both stabilizing and destabilizing/denaturant aspects of osmolytes to uncover both sides of the coin.

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Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117Effect of specific osmolyte depends on the solvent conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118Effect of osmolytes on protein destabilization is pH dependent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Destabilizing effects of osmolytes are concentration dependent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120Naturally occurring osmolytes that act as protein destabilizer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120Destabilizing effects of osmolytes on nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120Mechanistic models of solution dependent protein stabilization and destabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121Stabilization and/or destabilization by osmolytes modulates protein homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122Summary and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Introduction

Many plants, animals, and microorganisms have adapted toenvironmental stresses that normally denature macromolecules. Amechanism of adaptation that protects these cellular componentsagainst denaturation involves intracellular accumulation of smallorganic molecules known as osmolytes (Yancey et al., 1982; Yancey,2003, 2004). Stabilizing osmolytes are grouped into threemajor classes:polyols, amino acids and their derivatives, and methyl ammoniumcompounds (Fig. 1). Based on their effects on both stability and function

of proteins, they are often classified as compatible or counteracting.Compatible osmolytes increase protein stability against denaturationwith little or no effect on their function near room temperature (Pollardand Jones, 1979; Bowlus and Somero, 1979; Borowitzka and Brown,1974; Wang and Bolen, 1996; Wang et al., 1995). Counteractingosmolytes, on the other hand, possess special ability to protectintracellular proteins against inactivation and/or destabilization (Linand Timasheff, 1994; Yancey and Somero, 1980; Somero, 1986; Wangand Bolen, 1997; Baskakov and Bolen, 1998; Baskakov et al., 1998).Examples of organs and/or animals, which use counteracting osmolytesto protect themselves from denaturing effects of high concentrations ofurea, are mammalian kidney, and cartilaginous fishes and coelacanth(Bagnasco et al., 1986; Garcia-Perez and Burg, 1990; Nakanishi et al.,

Fig. 1. Representative chemical structure of osmolyte compounds in each class: amino acids and derivatives, polyols, and methylamines. In each class, two examples are given.

118 L.R. Singh et al. / Life Sciences 88 (2011) 117–125

1993; Yancey, 1985). Protecting osmolytes stabilize proteins againstdenaturation without altering their functional activities (Taneja andAhmad, 1994; Anjum et al., 2000; Xie and Timasheff, 1997a,b,c; Santoroet al., 1992; Foord and Leatherbarrow, 1998; Kim et al., 2003;Myers andJakoby, 1975). Osmolytes also help to stabilize unstable proteins and/orrefold misfolded proteins (Leandro et al., 2001; Leandro and Gomes,2008; Singh et al., 2007).

Osmolytesmodulate protein functions bymanipulating their stabilityunder physiological conditions by increasing melting temperature (Tm)and Gibbs free energy change (ΔGD°) associated with the denaturationequilibrium (Nstate↔Dstate) to various extents (Taneja and Ahmad, 1994;Xie and Timasheff, 1997a,b,c; Kaushik and Bhat, 1998; Santoro et al.,1992; Kim et al., 2003). Although most of the studies are focused on thestabilizing effects of osmolytes on proteins, many of them havedestabilizing effects on protein structure, stability, and function(Table 1). This has undermined our understanding of how osmolytescan be utilized by cells/tissues both as stabilizing and destabilizing agentsdepending upon the functional nature of a protein under specific cellularconditions. Various physicochemical approaches have gained significantinsights of osmolytes' interaction with proteins under protein stabilizingconditions (Timasheff, 2002; Bolen and Baskakov, 2001; Niebuhr andKoch, 2005; Bennion and Daggett, 2003). A survey of literature showsthat, apart from the stabilizing aspects of an osmolyte, the other face of anosmolyte (protein destabilization aspect) also has many importantbiological implications (Natalello et al., 2009; Baptista et al., 2000; Özcanet al., 2006). However, lack of knowledge on the mechanistic insights ofprotein destabilization by cellular osmolytes is poorly understood. In thisarticle, we have discussed various solution properties of osmolytesystems that turn a macromolecular stabilizer to a potential destabilizer.We also raise new insights of the potential biological implications ofdestabilization of macromolecules by osmolytes.

Effect of specific osmolyte depends on the solvent conditions

It is known that intracellular osmolytes increase the thermody-namic stability of macromolecules (Lin and Timasheff, 1994; Arakawaet al., 1990; Lee and Timasheff, 1981; Timasheff, 1993). For example,1.0 M trehalose increases the stability of lysozyme by 8 °C, and sucroseincreases the stability of RNase-Aby 4 °C (Kimet al., 2003; Kaushik andBhat, 2003). With increasing concentrations of sarcosine (0–6.0 M),the Tm and ΔGD° (at 25 °C) values of the wild-type RNase-Sa areincreased by 13.4 °C and 3.7 kcal/mol, respectively (Xie and Timasheff,1997a). Based on our work as well as from other laboratories, it can beconcluded that the effect of the efficiency of various osmolyte additives

on protein solution is usually solution condition-dependent (Tanejaand Ahmad, 1994; Anjum et al., 2000; Singh et al., 2005, 2009; Knappet al., 1999; Obon et al., 1996; Coelho-Sampaio et al., 1994; Xie andTimasheff, 1997a,b;Wetlaufer and Xie, 1995;Meng et al., 2001; Haqueet al., 2005a,b; Rishi et al., 1998). Polyol osmolytes undergo a pH-dependent stabilization of proteins (Haque et al., 2005a,b). Forexample, almost all polyol osmolytes (sorbitol, xylitol, mannitol,glycerol, and adonitol) have no significant effect on protein stabilityunder physiological conditions but strongly stabilize proteins at lowerpH values. Furthermore, results indicate that polyols have differenteffects on both stable and unstable proteins (Haque et al., 2005a,b). Inagreementwith these observations, Timasheff and coworkers (Xie andTimasheff, 1997a,b) reported that sorbitol stabilizes RNase-A more atpH 2.0 than at higher pH 5.5. On the other hand, stabilizing effect ofsugar osmolytes is dependent not only on the size of the sugarmolecule but also on the pH of the protein solution; the larger the size,the more is the stabilization effect (Poddar et al., 2008).

Similar to other polyols, sugars (glucose, fructose, galactose,sucrose, raffinose and stachyose) are also found to have pH-dependent stabilizing effects (Poddar et al., 2008). Similarly anothersugar, trehalose has also been reported to have pH-dependentstabilizing effects (Kaushik and Bhat, 2003). The current notion ofthis pH-dependence of ΔGD° of proteins is explained in the light of thereport that different charge states of a protein affects hydrophobicity(Kuhn et al., 1995). In fact, protein hydrophobicity increases withdecreasing pH due to the protonation of COO− groups (Kuhn et al.,1995). Polyol osmolytes, therefore, are more preferentially excludedfrom the hydrophobic surfaces by solvophobic interactions betweenthe hydrophobic- and the OH-groups present in polyol osmolytes(Kaushik and Bhat, 2003). Another interesting observation has beenmade that amino acid osmolytes (glycine, proline, isoleucine, leucine,phenylalanine, and valine) and amino acid derivatives (taurine and β-alanine) do not show significant pH-dependent stabilization effectson proteins in terms of ΔGD° (Taneja and Ahmad, 1994; Anjum et al.,2000 and unpublished results). Although they do not significantlyalter ΔGD° of proteins, they increase Tm at both neutral and lower pHvalues. These results indicate that the effect of amino acids and theirderivatives on proteins having different stabilities is indifferent interms of ΔGD°. Thus, the effect of amino acids and its derivatives issomewhat different from that of polyols and sugars.

Since both preferential hydration (and preferential exclusion) andpreferential binding act on a protein denaturation equilibrium, it isargued that ΔGD° of proteins is unperturbed in the presence of aminoacids and derivatives because there exists a perfect balance of the two

Table 1Destabilizing conditions of many proteins in the presence of osmolytes.

Protein Osmolytes Destabilizing conditions Type of study References

RNase-A TMAO Low pH Thermodynamic/structural Foglia (2008)Betaine High concentration Thermodynamic Knapp et al. (1999)

Lysozyme TMAO/betaine Low pH Thermodynamic Singh et al. (2005, 2009)Glycine/sarcosine/betaine Protein specific Thermodynamic Santoro et al. (1992)

GST-GFP Betaine High concentration Thermodynamic/structural Natalello et al. (2009)P39A Proline High concentration Structural Ignatova and Gierasch (2006)Prion protein TMAO Low pH Thermodynamic/structural Granata et al. (2006)Stem bromelain Trehalose/sucrose Protein specific Thermodynamic/structural Habib et al. (2007)Phosphofructokinase Glycerol Protein specific Thermodynamic Faber-Barata and Sola-Penna (2005)Lactate dehydrogenase TMAO Low pH Refolding Chilson and Chilson (2003)

Proline High concentrationCreatine kinase DMSO/glycine/proline High concentration Refolding Ou et al. (2002)

Glycerol High concentration Thermodynamic/structural Meng et al. (2004)Chymotrypsin TMAO Protein specific Thermodynamic/structural Kumar et al. (2005)Glucose dehydrogenase Sorbitol Protein specific Thermodynamic/refolding Obon et al. (1996)α-Crystallin Trehalose High concentration Thermodynamic Viner and Clegg (2001)DnaK+GroEL Trehalose High concentration Thermodynamic/kinetic Diamant et al. (2001)Cutinase Trehalose Higher pH Thermodynamic/kinetic Baptista et al. (2000)Cutinase Trehalose High concentration Unfolding/kinetic Melo et al. (2003)Native catalase Glycerol Higher pH Thermodynamic Costa et al. (2002)Glycogen phosphorylase b Proline Low concentration Thermodynamic Eronina et al. (2009)α-Lactalbumin Xylitol/inositol Low concentration Thermodynamic/structural Romero et al. (2009)α-1-Antitrypsin Sarcosine/betaine High concentration Thermodynamic/refolding Chow et al. (2001)Luciferase Sucrose/sorbitol/proline Protein specific Thermodynamic/structural Mehrabi et al. (2008)Ferricytochrome c Stachyose Protein specific Sedimentation Weatherly and Pielak (2001)

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opposing forces acting on the equilibrium (Jamal et al., 2009). Someosmolytes, despite of overwhelming protein stabilization near neutralpH, strongly destabilize proteins at lower pH values. Most surpris-ingly, the potent protein stabilizer, TMAO behaves as a denaturant atlower pH values (pH 5.0 and lower). Another methylamine osmolyte,glycine betaine also destabilizes or loses its stabilization power atlower pH values (pH 5.0 and lower) whereas near neutral pH, itbehaves as a strong protein stabilizer (Singh et al., 2009). A list ofosmolytes that destabilize specific proteins at physiological pH issummarized in Table 1. Apparently, our long standing belief thatcompatible osmolytes are protein stabilizing compounds is notuniversally true. Rather both stabilization and destabilization appearto be different faces of the same osmolyte. It is, therefore, highlyimportant to understand destabilizing condition(s) under which astabilizing osmolyte behaves as a protein denaturant and also thebiological importance of destabilization phenomenon.

Effect of osmolytes on protein destabilization is pH dependent

TMAO is a compound that has a pKa in the pH range of 4.56–4.75(Lin and Timasheff, 1994; Youxing and Bolen, 2003). Therefore,depending on the pH of the medium, this compound exists in azwitterionic (neutral) or positively charged form. It is well known thatthe zwitterionic form of TMAO is an excellent protein stabilizer andalso an efficient protein refolder (Lin and Timasheff, 1994; Yancey andSomero, 1980; Wang and Bolen, 1997; Baskakov and Bolen, 1998;Baskakov et al., 1998). In a systematic investigation, our laboratorydiscovered that the effect of TMAO is apparently pKa dependent.TMAO at pH above its pKa (pH≥5.0) stabilizes proteins while at pHbelow its pKa (b5.0), this compound behaves as a denaturant for allthree proteins studied (lysozyme, RNase-A and apo-α-lactalbumin).Chilson and Chilson (2003) have also shown that TMAO failed torefold acid and guanidinium chloride (GdmCl) denatured lactatedehydrogenase at pH 2.3. TMAO was reported not only to reducethermal stability of PrP at low pH but also to act as a denaturant atroom temperature (Granata et al., 2006). This destabilization is due tothe capability of the cationic form of TMAO to interact with theprotein backbone as well as to weaken the electrostatic interactions,which are important for the folding of PrP.

Vecchio's laboratory (Foglia et al., 2008) has carefully carried outan evaluation of thermal stability and secondary structure of RNase-Ain the presence and absence of TMAO at pH 7.0 and 4.0. Calorimetricand CD results show that TMAO stabilizes RNase-A at pH 7.0 but failsto do so at pH 4.0. Protein destabilization by TMAO may havebiological relevance in vivo. Indeed, there are many marine animalsthat are bottom dwellers and use TMAO as an osmolyte to counteractthe deleterious effects of pressure on proteins (Kelly and Yancey,1999). These animals also have H+ pump at their cell membrane tomaintain (near neutral) pH homeostasis (Claiborne et al., 2002). Oneobvious reason for having this H+ pump is that TMAO can protect thecellular proteins and enzymes against pressure denaturation onlywhen the pH is above the pKa of the osmolyte. The compatibleosmolyte glycine betaine is the most efficient osmoprotectant andbest excluder from the protein surface (Record et al., 1998; Courtenayet al., 2000). It reverses protein aggregation (Galinski and Trüper,1994), corrects mutant protein defects (Galinski and Trüper, 1994),and counters the harmful effects of urea and salts in vivo and in vitro(Burg and Peters, 1998; Coelho-Sampaio et al., 1994). Glycine betaine(in the pH range of 6.0–8.0) also serves as a potent stabilizer of manyproteins (Yancey and Somero, 1980; Anjum et al., 2000; Santoro et al.,1992; Singh et al., 2009). However, we have recently discovered thatat lower pH values (N5.0), glycine betaine has no effect on the stabilityof lysozyme at pH values in the range of 3.5–2.0 (both ΔGD° and Tmremain unchanged), and its effect on α-LA at pH values 4.5 and 4.0 isdestabilizing (both Tm and ΔGD° decrease with an increase in [glycinebetaine]) (Singh et al., 2009). Quite surprisingly, its effect on RNase-Aat a pH below 5.0 is more stabilizing than that at higher pH values. Inaddition, a normalized plot of the effect of pH on the stabilizing powerof glycine betaine on proteins indicates that this power increases witha decrease in pH of RNase-A solutions, whereas in cases of lysozymeand α-LA it is less effective at lower pH than that observed at pH 7.0(Singh et al., 2009).

Glycine betaine is not only a destabilizer at lower pH values butalso a denaturant to specific proteins at physiological pH. Recently, ithas been reported that green fluorescent protein (GFP) is destabilizedandmisfolded upon addition of glycine betaine (Natalello et al., 2009).Glycine betaine is also a structural destabilizer of a wide range ofproteins (Table 1). The protein structural destabilization by glycinebetaine was explained by the balance of two different interactions

120 L.R. Singh et al. / Life Sciences 88 (2011) 117–125

(favorable and unfavorable) (Bolen and Baskakov, 2001; Bolen, 2004;Ignatova and Gierasch, 2007; Auton and Bolen, 2005). In fact, theinteraction of betaine with specific amino acid side chains, such as Pheand Trp, (Auton and Bolen, 2005) seems to overcome the stabilizingosmophobic effect/preferential hydration (Bolen and Baskakov,2001), leading to structural destabilization. Furthermore, due to itspolar structure, betaine could also screen charge interactions,preventing the stabilization by salt bridges with consequent destabi-lization of the protein structure (Natalello et al., 2009). Anotherpossible explanation for a specific destabilization of protein by glycinebetaine is that glycine betaine acts as a specific destabilizing ligand formany proteins (Schiefner et al., 2004). Glycine betaine is also highlydestabilizing to liposomes containing nonbilayer lipids. The destabi-lizing effect of glycine betaine is more pronounced on membranescontaining the nonbilayer galactolipid, monogalactosyldiacylglycerolthan that on membranes containing the nonbilayer phospholipidssuch as phosphatidylethanolamine (Hincha, 2006). All these pieces ofevidence suggest that glycine betaine does not act to stabilize allbiomolecules but rather appears to be selective in nature.

Destabilizing effects of osmolytes are concentration dependent

At high concentrations many osmolytes destabilize or tend todestabilize proteins in general. Santoro et al. (1992) evaluated theeffect of various concentrations of osmolytes (glycine, sarcosine anddimethylglycine) up to their solubility limits on the stability of RNase-A and lysozyme. Each osmolyte exhibits a similar trend of approach-ing an optimum temperature for stabilization. However, upon reach-ing a maximum Tm, further addition of osmolyte results in a decreasein Tm. Such behavior suggests a tendency of osmolytes to destabilizeprotein at very high concentration. Knapp et al. (1999) have shownthat glycine betaine stabilizes RNase-A only at concentrations lessthan 3.0 M, and beyond this concentration, the osmolyte fails tostabilize it. In another observation, high concentration of glycinebetaine was found to destabilize model membranes under stressconditions (Hincha, 2006). Osmolytes have also been found tomodulate activity of molecular chaperones (heat-shock proteins) ina concentration-dependent manner suggesting a link between thechemical and molecular chaperones in regulation of protein folding invivo. Recent study shows that low concentrations of proline, glycerol,and glycine betaine increase activity of molecular chaperones, GroEL,DnaK, and ClpB thereby the yield of the native target proteins, likelydue to the promotion of local refolding within the chaperone andstabilization of the product (Diamant et al., 2001). However, highconcentrations of osmolytes have negative effects in the chaperonenetworks, as high viscosity affects the dynamics of chaperone–substrate interactions (Diamant et al., 2001).

Furthermore, less than 0.5 M glycine and 1.0 M proline improvedthe in vitro refolding yields of creatine kinase (Knapp et al., 1999;Obon et al., 1996), whereas at higher concentrations, this recoverywas compromised. Based on the structural and thermal stabilities ofbovine α-lactalbumin in buffer and dilute aqueous solutions oferythritol, xylitol, sorbitol, inositol, and glucose, Romero et al.(2003) reported that at low concentrations there was no structurestabilizing effect whereas at higher concentrations, all of them fairlystabilize the native protein conformation. It is interesting to note thatcells can accumulate osmolytes at concentrations up to severalhundred millimolar (Cayley et al., 1992). Therefore, it is very likelythat cells regulate many biological processes (especially proteinfolding, protein disaggregation, and protein–protein interactions) viaconcentration-dependent accumulation of osmolytes. However, cellsthat are not evolved with this property must have a system to pull outexcess osmolytes from the cellular pool. It may be for this obviousreason that, trehalose at high concentrations is toxic to macromole-cules and hence cells alleviate trehalose breakdown when level oftrehalose is increased from a threshold level (Diamant et al., 2001).

Naturally occurring osmolytes that act as protein destabilizer

Stability of all enzymes and proteins in cells is fine tuned.Therefore, increase in their thermodynamic stability is not the onlyimportant requirement of a cell under any specific stress condition.For, often times, organisms need systems that can give genericdestabilization of proteins under many circumstances such as thepresence of high concentration of salts or protein aggregatingconditions. Although compatible osmolytes are largely accumulatedto stabilize proteins and enzymes, nature has not ignored the use ofnon-compatible osmolyte system to give generic destabilization tothese macromolecules. Some common non-compatible osmolytesthat organisms use are arginine, lysine and histidine (Yancey, 2003). Ithas been reported that the non-compatible osmolyte, argininedestabilizes cytochrome-c and RNase-A (Bowlus and Somero, 1979;Taneja and Ahmad, 1994). It is also well known that the proteindestabilizing effect of arginine is due to its high preferential bindingpotential to proteins (Arakawa and Timasheff, 1983). We have earlierstudied the effects of all non-compatible osmolytes on the thermo-dynamic stability of various proteins, and reported that proteindestabilizing effects of arginine and histidine appear to be universallytrue (Rishi et al., 1998). We have also investigated the effect of pH onthe stabilization or destabilization of proteins by the non-compatibleosmolytes (Rishi et al., 1998). It has been observed that thedestabilization effect of arginine decreases non-linearly with decreas-ing pH. In fact, the destabilizing effect of arginine on RNase-A isreversed at pH values below 3.0, making it a protein stabilizer. Sincechange in pH condition shifts the denaturation equilibrium, ourresults further support that the destabilizing effect of arginine is dueto its binding to the denatured molecules. Both preferential hydrationeffect (surface tension effect) and preferential binding effect act onany protein denaturation equilibrium. At lower pH values preferentialhydration effect overcomes the preferential binding effect leading tostabilization of proteins. Thus, similar to the compatible osmolytes,that have two faces to modulate protein stability, non-compatibleosmolytes also have two different faces (protein stabilization anddestabilization).

Destabilizing effects of osmolytes on nucleic acids

Proline and glycine betaine are two major known osmoprotec-tants, which accumulate in plants, bacteria, algae, and marineinvertebrates in response to an array of abiotic stresses; mostprominent being the salinity stress (Measures, 1975; Csonka, 1989;Delauney et al., 1993). More often, the accumulation of theseosmolytes is the result of an adaptive de novo synthesis in cellscontributing amajor share among osmolytes (Boggess, 1976; Galinskeand Truper, 1982; Imhoff, 1986). In systematic studies of the effects ofa set of stabilizing osmolytes (proline, hydroxyproline, glycine,alanine, valine, leucine, serine, betaine, D-glucose, and sarcosine) onDNA stability, it was observed that osmolytes, proline and glycinebetaine destabilize double helix and lower the Tm of DNA in aconcentration-dependent manner (Rajendrakumar et al., 1997; Reeset al., 1993). None of the amino acid osmolytes tested could induceany stabilizing effect even at high concentrations. However, alanine,valine, leucine, and sarcosine could not greatly alter the Tm(Rajendrakumar et al., 1997). Hydroxyproline at its maximumaqueous solubility point (2.0 M) reduces Tm by 8.0 °C. Proline(1.0 M) reduces Tm of calf thymus DNA by 6.0 °C. Betaine at a similarconcentration reduces Tm of poly (dG-dC) by 5.0 °C and that of thebacterial DNA by 4.0 °C (Rees et al., 1993). In another development,proline (1.0 M)was observed to individually counter the effect of NaCl(0.5 M) and spermidine (10 mM) on DNA stability (Rajendrakumaret al., 1997). On the contrary, the co-addition of glycine (2.0 M) withsodium chloride (0.5 M) did not influence the effect of the latter onDNA, indicating the ineffectiveness of glycine in counteracting the salt

121L.R. Singh et al. / Life Sciences 88 (2011) 117–125

effect (Rajendrakumar et al., 1997). These observations highlight theimportance of proline or betaine to prevent the NaCl stress.

DNA destabilization by osmolytes has important cellular con-sequences. Several studies indicate that both in vitro bindingaffinities and rate of binding of certain transcriptional regulatoryproteins to their target sites on DNA are extremely sensitive to theelectrolyte concentrations of the buffers used (Record et al., 1985).Since DNA at physiological pH exists as a highly charged anion, it isexpected to be surrounded by cations which have a natural bindingaffinity. Moreover, the salts which accumulate during salinity stressmay also unduly stabilize the double helix, which could adverselyinhibit the DNA function in replication and transcription (Csonka,1989). Presumably, a mixture of proline (or betaine) and NaClcounterbalances each other such that the DNA stability is optimal. Inagreement, E. coli cells grown at very high salinity conditions (1.0 MNaCl) actively concentrate glycine betaine as much as 105 times thatof the medium (Rudulier et al., 1984). It has further been envisagedthat during severe stress conditions in bacteria, cellular constituentsmay completely be bathed in osmoprotectants that reach concen-trations above 1.0 M and interact with biomacromolecules (Rudulieret al., 1984). Similarly, the presence of high internal concentrationsof betaine under the stress-adapted conditions is found to reversethe effects of salinity mediated osmotic stress on DNA replicationand cell division in E. coli, which supports the role of osmoprotec-tants in alleviating the stress effects on DNA function (Meury, 1988).Thus, the selective accumulation of these two osmolytes (prolineand glycine betaine) in a wide range of organisms under the saltstress appears to be a conserved adaptive measure rather than amere coincidence. One can, therefore, speculate that if the effects ofosmolytes are to enhance only the macromolecular stability, itwould have been impossible for the cells to counter the effect of saltson nucleic acids.

Not only DNA, osmolytes also destabilize RNA. Lambert and Draper(2007) studied effects of nine osmolytes on the secondary and tertiarystructures of six RNA structures differing in stability and complexity.The osmolytes used were selected to be representative of differentchemical classes of naturally occurring osmolytes, amino acids(proline and betaine), methylamines (betaine and TMAO), andpolyols and sugars (sorbitol, sucrose, glycerol, ethylene glycol, andmethanol). All the osmolytes destabilize RNA secondary structure,although to different extents, probably because they favor solubiliza-tion of base surfaces. Osmolyte effects on tertiary structure, however,can be either stabilizing or destabilizing. Urea and proline areuniformly destabilizing. There is considerable variation among RNAsin the effectiveness of an osmolyte. For example, the U1061A andGACG variants of the rRNA fragment, which differ by only fournucleotides in sequence, differ by nearly a factor of two in sensitivityto methanol and ethylene glycol (Lambert and Draper, 2007). Thedifferential effect of a given osmolyte on the secondary and tertiarystructures of RNAwas explained on the basis of preferential hydrationeffect. Because these osmolytes favor burial of RNA backbone, i.e.,favorable osmolyte interactions with base surfaces must be out-weighed by unfavorable interactions with backbone.

Mechanistic models of solution dependent protein stabilizationand destabilization

The experimental evidences describing protein interaction withosmolytes (preferential interaction measurements) discussed earlierwere obtained at or near the physiological pH only. In the absence ofthe experimentally derived protein–osmolyte preferential interactionparameters at low pH, one can easily obtain the parameters usingWyman linkage relation (Wayman, 1964). Briefly, upon changing onlythe osmolyte concentration, the change in the equilibrium constant ofthe two-state denaturation (Nstate↔Dstate) is given by the differencein the number of osmolyte molecules bound by the denatured (νD)

and native (νN) protein molecules, as shown in the followingequation:

δ lnKBD = δln a = Δν = νD−νN

where KD° is the equilibrium constant of protein denaturation at 25 °C;a, the thermodynamic activity of the osmolytes; and Δν, thedifference between the number of molecules of the osmolyte boundper mole of protein between the denatured (νD) and native (νN)states. Furthermore, if Δν, the value of the slope of the plot of lnKD°versus lna, is negative, the preferential exclusion effect dominatesover the preferential binding effect, and if Δν is positive, thepreferential binding effect dominates over the preferential exclusioneffect. Using Wyman linkage relation, we for the first timedemonstrated the difference in the binding of the osmolyte, TMAOat protein stabilizing and destabilizing conditions (Singh et al., 2005).The evaluated slope (Δν) is negative at pH values of ≥5.0 (proteinstabilizing condition by TMAO), suggesting that TMAO is preferen-tially excluded from the domains of RNase-A, lysozyme, and apo-α-lactalbumin (α-LA). On the other hand, for all proteins, the slope (Δν)is positive at pH values b5.0 (protein destabilizing condition byTMAO), suggesting that the effect of the binding of TMAO with sidechains dominates over the effect of the exclusion of TMAO from thepeptide backbone. In another observation, Kaushik and Bhat (2003)reported that estimated Δν of trehalose in RNase-A solution becomesmore and more negative with decreasing pH. Interestingly, a decreasein the pH of the protein solutions also results in an increase in ΔTm inthe presence of trehalose. Taken together, preferential binding ofosmolytes at lower pH or high concentration of osmolytes is the causeof destabilization of proteins by osmolytes.

It is well known that the stabilizing osmolytes are preferentiallyexcluded from the immediate vicinity of the protein surface(Timasheff, 2002; Arakawa et al., 1990), and this exclusion impliesa solvophobic interaction between groups (peptide backbone andside chains) on the protein surface and the protecting osmolytespecies. It has been shown (Timasheff, 2002; Arakawa et al., 1990)that there exist two opposing forces between the protein and thestabilizing osmolyte acting on the denaturation equilibrium, native(Nstate)↔denatured (Dstate). The preferential exclusion of theosmolyte from the protein domain, which originates from theunfavorable interaction between the osmolyte and peptide back-bone, favors N state, whereas the preferential binding of theosmolyte with the protein, which originates from the favorableinteraction between the osmolyte and protein side chains, favors Dstate. The observed ΔGD°, the free energy change for proteinunfolding, therefore, results from the net differences betweenthese two opposing interactions (Fig. 2).

The critical factor is the partitioning between water and osmolyteat solvent-exposed surfaces of a protein. While stabilizing osmolyteshave an overwhelming tendency to be excluded from the proteinsurface, forcing the polypeptide to adopt a compactly folded structurewith a minimum of exposed surface area, denaturing osmolytesaccumulate or bind at the surface and promote unfolding (Singh et al.,2005, 2009; Bolen and Baskakov, 2001; Arakawa et al., 1990;Courtenay et al., 2000, 2001; Felitsky et al., 2004; Timasheff, 1992).Accumulation or exclusion occurs primarily at polar surfaces,particularly the backbone amides (Auton and Bolen, 2005; Courtenayet al., 2001). Thus, the sensitivity of a protein to osmolytes dependsprimarily on the degree to which its polar peptide backbone becomesburied upon folding into the native structure and the relative strengthof osmolyte versuswater interactions with peptide groups (Auton andBolen, 2005). Therefore, protein stabilization by osmolytes at certainsolvent conditions is due to solution conditions favoring more andmore exclusion of osmolytes from the protein surface. On the otherhand, certain solution conditions disfavor exclusion that consequentlyleads to binding and destabilization. For the methyl group containing

Fig. 2. Schematic diagram of the preferential binding and exclusion exhibited byosmolytes. (a) Protein in the absence of osmolyte. (b) Osmolyte is preferentiallyexcluded from the protein domain, i.e. protein is preferentially hydrated, leading tostabilization of the native protein. (c) Osmolyte is preferentially bound to the proteinleading to the stabilization of the denatured protein. The blue circle around N and Dprotein molecules shows hydration layer whereas red filled circles indicate osmolyte.The net effect of the osmolyte on the protein stability (ΔGD°) is the result of the finebalance between preferential exclusion (b) and preferential binding (c) of theosmolyte.

Fig. 3. A model showing effects of osmolyte on a protein. Under stabilizing conditions,an osmolyte can force the protein (a) into more stable (folded) conformation (native-like species) (c), whereas under destabilizing conditions, it may turn into less stable(unfolded) conformation (b), which may be prone to degradation under cellularconditions. The size and direction of arrows indicate the shift in equilibrium betweenrespective conformations.

122 L.R. Singh et al. / Life Sciences 88 (2011) 117–125

osmolytes, the binding is mediated by its methyl moiety (Lin andTimasheff, 1994; Santoro et al., 1992). In addition, low pH alsoenhances hydrophobicity (Kuhn et al., 1995) and thus augmentspreferential binding (Singh et al., 2005; Kaushik and Bhat, 2003). Onthe other hand, the destabilization effect of osmolytes on proteins attheir high concentrations may most probably be due to change in theproperty of osmolytes at high concentrations (Timasheff, 1992;Samuel et al., 2000; Sonoda and Skaf, 2007). It has been shown thatosmolytes at high concentrations form clusters (Samuel et al., 2000;Sonoda and Skaf, 2007; Lerbret et al., 2005). The molecular detail ofdestabilization by these clusters may be that the cluster promotes

reduction in the backbone surface and increase in the side chaininteractions.

Stabilization and/or destabilization by osmolytes modulatesprotein homeostasis

Achieving the correct balance between folding and degradation ofmisfolded/unstable proteins is critical for cell viability. In cells, thequality of newly synthesized proteins is monitored in regard to properfolding and correct assembly in the early secretory pathway, thecytosol and the nucleoplasm. Proteins recognized as non-native/unstable in the cell are removed and degraded by proteosome (Rothet al., 2008). Failure to do so leads to proteopathy (abnormality inprotein homeostasis). Since stabilizing osmolytes are capable ofmonitoring misfolded/unstable proteins and direct them to the foldedstate, they will increase homeostasis of certain unstable proteins.Destabilizing osmolytes, on the other hand, are like denaturantswhich will destabilize proteins and consequently, help to promotedegradation of certain overtly stable or aggregated proteins present inthe cell. Such a scenario is shown in a hypothetical model in Fig. 3. It is,therefore, logical that certain osmolytes (stabilizing or destabilizing)are involved in regulation of protein homeostasis.

Various studies have indicated the importance of osmolytes inregulating protein homeostasis (Leandro and Gomes, 2008; Singhet al., 2007;Welch, 1996; Brown et al., 1996, 1997; Tatzelt et al., 1996;Eleutherio et al., 1998; Edington et al., 1989; Andrew et al., 2003).These osmolytes, in fact, can rescue function to many misfoldedproteins by refolding and preventing degradative attack fromproteolytic enzymes. Changes in protein stability can also be broughtabout simply by changing the property of the accumulating osmolytesin the cell. For instance, betaine, a stabilizing osmolyte at lowconcentrations behaves as a denaturant at high concentrations(Natalello et al., 2009). In fact, almost all age related diseases andmany genetic diseases are due to problems of protein homeostasiseither due to promotion of proteosome-mediated degradation orformation of aggregates (Powers et al., 2009). All these diseases aretogether called protein conformational diseases. This group ofdiseases include Alzheimer's disease, transmissible spongiformencephalitis, serpin deficient disorders, haemolytic anaemia, Hun-tington disease, cystic fibrosis, diabetes type II, amylotropic lateralsclerosis, Parkinson's disease, dialysis related amyloidosis and more

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than 15 other less known diseases (Soto, 2001). In theory, treatmentwith appropriate proteostatic modulators could reverse these proteinfolding defects. Since destabilizing osmolytes are like denaturants,one could easily speculate that these osmolytes will be potentiallyuseful for removing protein aggregates produced by mutant proteins.In agreement, destabilizing osmolytes like arginine and lysine arecommonly used to solubilize inclusion bodies and insoluble proteinaggregates (Das et al., 2007; Chen et al., 2008; Bajorunaite et al.,2007).

On the other hand, stabilizing osmolytes are excellent proteinrefolders. Therefore these osmolytes will be useful in rescuingfunctions of mutant proteins, which is due to instability or enhancedproteosomal mediated degradation. Recently, it has been shown thatspecific osmolytes are able to correct suchmisfolded conformations toprevent the excessive degradation and consequently promote theintracellular functional activity of the mutant proteins (Baskakov andBolen, 1998; Welch, 1996; Brown et al., 1996, 1997; Tatzelt et al.,1996; Eleutherio et al., 1998; Edington et al., 1989; Andrew et al.,2003). For instance, proline protected chicken liver fatty acid synthase(Park et al., 2002), chicken egg lysozyme (Samuel et al., 2000), andrabbit skeletal muscle creatine phosphokinase (Meng et al., 2001)against aggregation. A wide range of osmolytes can restore mutantprotein folding defects in cystathionine beta synthase (Singh et al.,2007) and phenylalanine hydroxylase (Leandro et al., 2001).However, other researchers reported that osmolytes rather potentiatemisfolding or have no effect on protein aggregation. For example,TMAO promoted aggregation during refolding of RNase (Ratnaparkhiand Varadarajan, 2001). Glycine even at high concentrations (1.8 M)caused minor influence on chicken liver fatty acid synthase aggrega-tion (Park et al., 2002), and it did not inhibit protein aggregationduring refolding of chicken egg lysozyme (Samuel et al., 2000).

Summary and perspectives

It is well known that solvent conditions or osmolyte concentra-tions can easily alter protein stability or folding. Depending uponconcentrations and the solvent conditions, naturally occurringosmolytes can have stabilizing or destabilizing effects on proteins.Thus, it is important to look into both stabilizing and destabilizingaspects of osmolytes to determine their precise role under physio-logical conditions. An osmolyte may even act as a ligand to specificproteins to regulate protein stability. Therefore, judicious use ofosmolytes to disaggregate or refold mutant proteins must besystematically carried out according to the property that the osmolytepossess or by making appropriate co-solvent conditions. Stabilizingosmolytes in particular will be useful to refold proteins and thedestabilizing osmolytes must be used appropriately to form disag-gregates from aggregated proteins. Possible involvement of osmolytesin modulating protein homeostasis must be extensively investigated.Nevertheless, osmolytes can be developed as potential future tools atthe level of therapeutic interventions aimed at tackling proteins withmisfolded/aggregated conformations, a major pathological conditionin a number of genetic diseases. The prospect of using naturalosmolyte as a therapeutic tool for neurodegenerative diseases inparticular appears to be quite exciting without fear of major sideeffects. However, while these ideas appear to be quite promising,more studies are needed to validate their effectiveness as a potentialtherapeutic target. Understanding proper mechanism of action ofosmolytes in these pathological conditions can have far reachingconsequences in developing better therapeutic tools for the preven-tion and/or management of such diseases. The current discrepancy isdue to the fact that earlier we did not have a clear picture about theproperties of the stabilizing and destabilizing osmolytes and alsoconditions in which an osmolyte will fall as stabilizing or destabilizingagent. Our long standing belief that all osmolytes apparently aremacromolecular stabilizers is no longer valid. Therefore, appropriate

use of destabilizing or stabilizing osmolyte depending on the type ofprotein misfoldedness (either unstable or aggregate) will resolvethese inconsistencies about osmolyte-induced mutant protein refold-ing or resolubilization of aggregation.

Acknowledgements

FA thanks the Department of Science and Technology, India andthe Council of Scientific and Industrial Research, India for theirfinancial support.

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