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Role of naturally occurring osmolytes in protein folding and stability
Archives of Biochemistry and Biophysics 491 (2009) 1–6
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Review
Role of naturally occurring osmolytes in protein folding and stability Raj Kumar * Department of Basic Sciences, The Commonwealth Medical College, 501 Madison Avenue, Suite 205, Scranton, PA 18510, USA
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Article history: Received 29 August 2009 and in revised form 14 September 2009 Available online 19 September 2009 Keywords: Osmolyte Protein folding Protein structure Cooperativity Protein backbone Amino acid side chain
a b s t r a c t Osmolytes are typically accumulated in the intracellular environment at relatively high concentrations when cells/tissues are subjected to stress conditions. Osmolytes are common in a variety of organisms, including microorganisms, plants, and animals. They enhance thermodynamic stability of proteins by providing natively folded conformations without perturbing other cellular processes. By burying the backbone into the core of folded proteins, osmolytes can provide significant stability to proteins. Two properties of osmolytes are particularly important: (i) their ability to impart increased thermodynamic stability to folded proteins; and (ii) their compatibility in the intracellular environment at high concentrations. Under physiological conditions, the cellular compositions of osmolytes may vary significantly. This may lead to different protein folding pathways utilized in cells depending upon the intracellular environment. Proper understanding of the role of osmolytes in cell regulation should allow predicting the action of osmolytes on macromolecular interactions in stressed and crowded environments typical of cellular conditions. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Proteins need to maintain their natively folded structures for proper functions under physiological conditions [1]. However, most proteins are sensitive to changes in cellular and environmental conditions including temperature, pressure, and the presence of salts and other solutes [2–4]. Therefore, under physiological conditions, proteins require to counter-balance any significant perturbations in these thermodynamic conditions to avoid significant changes in their secondary and tertiary structures [5,6]. Protein failing to adapt to such conditions may result in a partial or complete loss of their functional activity [2–4]. In order to adapt to such perturbations due to extreme conditions, nature has created certain mechanisms such as accumulation of small organic solutes, also known as ‘‘osmolytes” [5–7]. Osmolytes are naturally occurring small organic molecules often referred to as ‘‘chemical chaperones”, and are typically accumulated in the intracellular environment at relatively high concentrations that can increase thermodynamic stability of folded proteins without perturbing other cellular processes [4,7–10]. In many cases, the environmental stress due to harsh denaturation conditions, threatens the stability of intracellular macromolecules [2–4,11,12]. Therefore, many osmolytes have been selected for their ability to stabilize cell components such as proteins [13]. These naturally occurring compounds exert a force to fold proteins that are highly unstructured in an aqueous environment [14,10,15]. It has been demonstrated * Fax: +1 570 504 9660. E-mail address: [email protected]. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.09.007
that osmolytes can cause proteins to fold cooperatively into native-like, functional species [16–19]. It has been shown that the protein backbone is effectively osmophobic, and osmolytes can provide significant stability to protein by hiding the backbone into the core of folded proteins [20–22]. The ability of osmolyte to increase the driving force for protein folding is due to a solvophobic effect on the peptide backbone exposed in the unfolded state [23–25]. Another feature of these molecules is that they make the unfolded state of protein in osmolyte solution very unfavorable relative to the folded state, without changing the rules of protein folding that occur in dilute solutions [26,27]. It is generally acknowledged that the functions carried out by proteins depend upon their structures [1]. It is assumed, based on the pioneering work of Anfinsen and coworkers that the primary sequence of a protein contains all the information required for it to fold properly [28]. However, it is not yet possible to predict with certainty the folded form of a protein from its primary sequence [1,6]. Since the peptide chains of all proteins have an identical composition, how do the side chains dictate overall folding? Also because of an astronomically large number of possible polypeptide chain conformations, how does a given sequence find its specific native structure in a finite time? There are suggestions that once proteins are synthesized, the highly disordered unfolded state pass through well-defined partially structured transition states, to fully folded forms (Fig. 1) [29]. After synthesis on the ribosome, protein is assumed to fold in the endoplasmic reticulum, aided by molecular chaperones that deter aggregation of incompletely folded species [29]. The correctly folded protein is secreted from the cell and functions normally in its extracellular environment
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pared with the buffer [52]. On the other hand, same is not true for another protease, chymotrypsin [52], suggesting that effects of osmolytes on the protein folding/stability are unique in nature and could vary [26]. Thus osmolytes may be an answer to such problems associated with protein stability/folding [7,26]. In this review article, we have summarized an up dated knowledge of presence of various organic osmolytes in nature, and their role in maintaining/inducing folding and/or stability of macromolecules that could lead to better management of various cellular and physiological functions of proteins.
Osmolytes in nature
Fig. 1. Schematic diagram of some of the states accessible to a polypeptide chain following its biosynthesis. There are suggestions that once proteins are synthesized, the highly disordered unfolded/unstructured state pass through well-defined partially structured/folded transition states (intermediate stage), to fully native folded and functionally active conformation (final stage). After synthesis on the ribosome, protein is assumed to fold in the endoplasmic reticulum, aided by molecular chaperones that deter aggregation of incompletely folded species. The correctly folded protein is secreted from the cell and functions normally in its extracellular environment. Under certain conditions protein unfolds, at least partially, and becomes prone to aggregation. This can result in the formation of fibrils or amyloids and other aggregates that accumulate in tissue/cell. It is likely that small aggregates as well as the highly organized fibrils and plaques, can give rise to pathological conditions, at least in some cases. Naturally occurring osmolytes could facilitate the intermediate reactions by shifting it to the final natively folded conformation, thereby making them less susceptible to aggregation. U, unfolded state; I, partially structured/folded intermediates; N, globular native form (based on Ref. [29]).
[29]. Intracellular proteins are also aided by several chaperone proteins to maintain their proper structure under physiological conditions [30,31]. Under certain conditions, protein unfolds, at least partially, and becomes prone to aggregation [32–35]. This can result in the formation of fibrils and other possible aggregates that accumulate in tissue [36–41]. It is likely that small aggregates as well as the highly organized fibrils and plaques, can give rise to pathological conditions, at least in some cases [42,43]. The question then is how the ensembles of unfolded or partially folded proteins are in such a small population to avoid aggregation in the biological environment. The answer to this possibly lies with the cooperative nature of protein folding process [16–19]. In the two-state model of protein folding, the partially folded intermediate states are small [44]. Cooperativity of protein folding process has long been documented in well-defined native proteins capable of carrying their functions [16–19]. It seems that cooperative nature of protein folding could be equally crucial for avoiding protein/ peptide aggregation [16–19]. In fact, several studies, including ours have shown that in the presence of certain osmolytes, long stretches of unstructured or intrinsically disordered regions/domains of proteins can be folded in a cooperative manner, and the resulting conformations are native-like with significant functional activities [22,44–50]. Many of these intrinsically disordered regions are found in the activation domains of various transcription factors, which are involved in the gene regulation [51]. There are some reports showing that even a relatively folded protein with an active globular structure can also adopt a more functionally active conformation in the presence of osmolyte [52]. We have earlier shown that in the presence of osmolyte, trimethylamine-Noxide (TMAO)1, trypsin, a proteolytic enzyme can adopt a conformation that facilitates its enzyme activity by several fold when com1 Abbreviations used: TMAO, trimethylamine-N-oxide; LEM, linear extrapolation method.
As discussed above, organic osmolytes are used widely in nature to protect cellular proteins against harsh conditions such as the effects of dehydrations, other hypertonic states, or the buildup of potentially denaturing metabolites [4,53–61]. Hyper-osmotic conditions set off a response in most cell types that turns on the synthesis of protective organic osmolytes [7]. There are three major chemical classes of protecting osmolytes, and while they all have varying abilities to fold/stabilize proteins, they also have different physical and chemical properties that may alter the function of a second osmolyte present in cells [4]. A list of some common categories of the osmolytes is shown in Fig. 2. The selection of osmolytes depends on the duration of the osmotic stress and the availability of substrates and osmolytes in the surroundings [7,26]. In deep sea invertebrates, high levels of several osmolytes are present [57]. To balance the high osmolality of sea water and to counter-balance high concentrations of urea, these animals accumulate organic osmolytes in their tissues and extracellular fluid, particularly in the elasmobranchs [4]. Of particular interests are the methylamines that accumulate with urea to counteract its denaturing effects [26]. Since the most important determinant of the difference between protein denaturant (e.g. urea) and osmolytes (e.g. methylamines) is their opposite effects on the configuration of protein backbone, therefore, at a particular ratio of methylamines to urea (generally 2:1), the denaturing effects of urea are counteracted [62]. Yeast and fungi are known to generate and/or accumulate different osmolytes from the environment [63,64]. Glycerol is accumulated in response to hyper-tonicity by increasing its production and reducing its efflux [63,64]. Besides several others, high levels of glycerol are employed as an osmolyte by Saccharomyces cerevisiae [63,64]. Several different organic osmolytes are reported to be accumulated in bacteria (particularly Escherichia coli and Salmonella typhimurium) in response to hypertonicity [65]. When growing in the absence of exogenous osmolytes, E. coli cells increase the synthesis of certain osmolytes e.g. trehalose [65]. Organic osmolytes generally are accumulated in plants by increasing the rate at which they are synthesized [66]. Plants subject to salt stress accumulate organic osmolytes in the cytoplasm of their cells [66]. The increased accumulation of osmolytes in leaves of stressed plants is partly regulated through changes in the gene expression of their biosynthetic enzymes [67]. However, the mechanism of action for these changes has not been well understood. The osmolality of mammalian blood is normally kept constant by a combination of thirst and varying urinary concentrations [19]. Therefore, most mammalian cells are not normally exposed to the extreme osmolalities experienced by several other kind of non-mammalian cells [4,19]. However, certain mammalian cells contain considerable concentrations of organic osmolytes [4,64]. Renal medullary cells are known to contain the highest levels of organic osmolytes as a consequence of being exposed to extremely high concentrations of salt and urea [4,19,64]. The mechanisms of accumulation and their regulation in response to high NaCl
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Fig. 2. Diagram showing two examples each in three major categories of osmolytes. There are a number of well-known naturally occurring osmolytes which fall into three chemical classes: methylamines, such as TMAO, Choline-O-sulphate, and sarcosine: polyols such as sorbitol, glycerol, sucrose, and trehalose; and certain amino acids, such as glycine, taurine, proline, and betaine. Each class interacts with both the peptide backbone and to varying extent, also with amino acid side chains. The balance of these effects determines the potency of the osmolyte to promote protein folding and solubility. Thermodynamic calculations and experimental results support the view that the powerful solvophobic effects of osmolytes on the peptide backbone dominate, such that the relative Gibbs free energy of the unfolded state is less favorable than that of the folded state.
and urea have been most thoroughly studied in renal medullary cells [64]. Aside from renal cells, the normal levels of organic osmolytes in specific cell types have neither been surveyed intensively nor have been examined in many tissues under pathophysiological conditions, but it has been established that in many cells/ tissues it may well reach in a very high concentration range [64]. It has been calculated that osmolyte concentrations in whole tissues may often reach in the range of 300–400 mmols/kg of cell water, meaning that in certain compartments, they are almost surely much higher [7]. Desert mouse kidney is one extreme case where the concentration of an osmolyte in this animal occurs there at 2.5 M, allowing retention of structure and function of essential proteins [4,7]. This is because in this case the urea concentration may reach up to 5 M [4,7]. Trimethylamine-N-oxide (TMAO), an osmolyte extensively studied for protein folding, is natural solute with high concentrations in the shark, where it exists to prevent the denaturant effect of the high urea levels in those animals [4,7,26]. In fact, it is common to find more than one intracellular organic osmolyte in cells [4,7]. Human kidney, for example, contains several osmolytes, including glycine betaine, sorbitol, inositol and taurine [4,7]. The presence of several osmolytes inside cells raises a number of questions about their roles in protecting intracellular macromolecules against the environmental stresses. The most immediate question is whether these osmolytes work individually in an independent manner and/or in conjunction with each other. If osmolytes produce their effects in combinations; are these effects synergistic in their ability to protect cellular components against potentially denaturation conditions that cells may encounter. Even in the same cell, there could be many different osmolytes present [4,7]. However, their concentrations and compositions may vary in different cell types [64]. Since the protection provided by an osmolyte does not depend on specific chemical interactions with the macromolecules, in principle any of the osmolytes should be capable of replacing each other, depending upon either endogenous or exogenous availability of particular osmolyte(s) [64]. There are some reports to support this idea [68–72]. For example, when renal cells in culture are maintained at a constantly high salt concentration, an induced change in an organic osmolyte is reciprocated by an opposite change in the amount of another one with similar protective effects [73]. This raises the question about the natural selection criteria for osmolytes in cells. Why do cells need so many osmolytes, and why are there so many classes of osmo-
lytes? Given their interchangeable protective effects, why should one osmolyte be preferred over another? The explanation may be that even though the protective effects of osmolytes are non-specific, an osmolyte or the mechanisms that regulate it may have specific effects. Bolen’s laboratory has recently shown that the effects of differing osmolytes are additive, so that a cell may contain even higher summative concentrations of these protein pro-folding molecules [74,75]. A hypothetical model of such actions of osmolytes on the folding of an unstructured protein is shown in Fig. 3. Of course, membrane-limited intracellular compartmentalization in the cell and the intracellular environment may also make for quite high concentrations in specific loci or compartments [74,75]. Therefore, it is logical that cells do not routinely express extremely high levels of osmolytes [26]. Several classes of well-known organic osmolytes are present in mammalian cells, where they function to protect proteins from denaturation under certain conditions [1–7]. Since, many cellular proteins function through interaction with other binding protein partners to form large assembly of protein complexes that must have enough flexible conformation(s) to include or exclude binding partners in response to specific cellular functions; it must not have too rigid structure [48,51]. Therefore, maintenance of excessive amounts of folded proteins would interfere with release of bound protein partners that require a structural relaxation, with the loss of conformation required for proteolysis and turnover and maintenance of normal osmotic, salt-based transmembrane gradients [20]. Therefore, it is possible that specific cells utilize specific osmolyte(s) to encourage optimal functioning conformation(s) of certain proteins [7]. This may in fact be true for several transcription factors, which possess intrinsically disordered activation domains, and have cell-specific effects in gene regulation [51]. Based on our findings with the intrinsically disordered activation domain of steroid receptors [51], it can be hypothesized that cell-specific effects of signals passed by steroids through their receptor to regulate the expression of specific target gene may be dependent in part to the presence of specific sets and concentrations of cellular milieu.
Osmolyte-induced protein folding Protein folding process is reversible in nature, and the equilibrium between unfolded and natively folded does not involve cova-
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Fig. 3. A model showing osmolyte-induced characteristics in thermodynamic terms of solvophobic effects. Osmolytes interact unfavorably with the unfolded or unstructured states, resulting in preferential depletion of osmolyte proximate to the protein surface, thereby forcing protein to adopt folded conformation(s). Protein is shown with green color. In the center, protein is intrinsically disordered or unstructured, and is surrounded by only water molecules (small blue filled circle). O1, O2, O3, or O4 (shown by large filled circle, red, green, blue, and purple, respectively) represent the presence of various osmolytes (Ox). In the presence of each osmolyte, due to sequestering of water molecules by osmolytes, protein adopts folded conformation, which may have similar or different overall fold depending upon the nature of osmolyte and protein. The final natively folded conformation of protein may be determined by the sum of these osmolytes. Effects of these osmolytes on the folding of protein may be additive in nature. Thus, the availability of number of osmolytes in a particular cell may decide the concentration of osmolytes required to fold protein into a functionally active conformation. The final outcome may also be determined by the nature of other interacting macromolecules and small ligands (if any).
lent bonds during inter-conversion between unfolded and natively folded conformations [20]. Therefore, a thermodynamic process of protein folding can be framed in terms of solvent interactions with the unfolded and native states [20]. Osmolytes push the folding equilibrium toward natively folded conformations, whereas denaturing solutes such as urea push the equilibrium toward unfolded conformations [20]. The protecting osmolytes raise the free-energy of the unfolded state, favoring the folded population, whereas denaturing solutes lower the free-energy of the unfolded state, favoring the unfolded population [20]. Accordingly, osmolytes interact unfavorably with the unfolded state, resulting in a preferential depletion of osmolyte proximate to the protein surface [20]. A model for such osmolyte-induced characteristics has been shown in thermodynamic terms (Fig. 4). However, there are certain limits to this phenomenon, and thus it cannot be termed as a universal theory that can account for the mechanism by which osmolytes interact with the protein to affect stability [20]. There are reports showing that the osmolytes predominantly affect the protein backbone, a common component to all polypeptide chains [26]. Though the magnitude of the energy of interactions with each peptide bond is very small, peptide bonds are by far the most numerous structural component of a protein. Consequently, the sum of such interactions can be quite large [20,75,26]. It is the balance between osmolyte-backbone interactions and amino acid side chain-solvent interactions that determines the outcome on protein folding [26]. Intrinsically disordered or unstructured regions do not of themselves contain sufficient hydrophobic amino acid side chains to fold spontaneously [51,76–80]. Thus, addition of an osmolyte tips the balance to a favorable negative free energy for folding [26]. One interesting aspect of osmolyte-induced protein folding is a very weak protein:osmolyte interaction, and therefore, the free-en-
Fig. 4. A schematic diagram showing protein stabilization brought about by the action of osmolyte. Osmolytes act by increasing DG between the natively folded (Nbuffer) and unfolded ensembles. DG1 is the unfolding Gibbs energy difference between native and unfolded (Ubuffer) protein in aqueous buffer solution, and DG3 is the Gibbs energy change for the same reaction in the presence of an osmolyte. Transfer of Nbuffer or Ubuffer from aqueous buffer to osmolyte solution (Nosmolyte and Uosmolyte, respectively) raises the DG2 of the unfolded ensemble much more than it does for the natively folded ensemble (DG4), resulting in a greater stability of the protein in the osmolyte solution than in aqueous solution (based on Ref. [44,62]).
ergy effects are additive because osmolytes do not occupy any significant fraction of the backbone surface [20,26]. This essentially allows no competition for backbone binding sites [26]. The natural protein folding force derives primarily from the composition and distribution of amino acid side chains [28]. In unstructured proteins, the hydrophobic amino acids are insufficient, relative to the charged side chains, to cause spontaneous
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folding [81–85]. Addition of the solvophobic effect through addition of an osmolyte can tip the free-energy balance in the favor of folding, as the backbone collapses to avoid solvent with osmolyte [26]. The quantity of osmolyte(s) required depends on both its inherent solvophobic effects in the interaction with peptide backbone, and the free-energy balance provided by the sum of all backbone-osmolyte interactions and the sum of all amino acid side chain-solvent interactions [74,75,26]. Because protein backbone comprises the most numerous functional groups in proteins, osmolyte-induced conformations are driven by very strong forces, and evidence from the several systems studied thus far indicates native folded functional species result [51,26]. Given the essential roles of osmolytes in protecting cells, it is important to determine how effect of one osmolyte on protein stability is affected by the presence of other osmolyte(s). One of the primary roles of the thermodynamic parameters is to attain predictive understanding of the energetic changes, which are responsible for folding/unfolding of proteins at the level of amino acids [26]. Major forces, which are fundamental to our understanding of protein folding and stability, can be attributed to the transfer of free-energies that accompanies the transfer of amino acid side chains and peptide backbones from water to other solvents, and thereby measuring the hydrophobicity and hydrogen bonding of proteins [86–88]. Since, it is well established that the peptide backbone makes the dominant contribution to the free-energy change between the native and denatured states, these thermodynamic approaches have been extensively used to determine osmolyte-induced protein folding [26]. Because organic osmolytes within the cellular milieu stabilize intracellular proteins to protect cells against denaturation conditions, a direct link between cellular function and energetics of protein folding becomes a central issue of solvent–protein interactions in vivo under physiological conditions [86–88]. Auton and Bolen recently demonstrated that free-energies of side chain and backbone transfer from water to osmolytes can be achieved by predicting solventdependent cooperative protein folding/unfolding free-energy changes (in terms of m values) [87]. An experimental measure of the effect of an osmolyte on protein stability can be determined from the m-value, obtained from the slope of a plot of the native to denatured free-energy change as a function of osmolyte concentration [74,75,26]. Since the mvalue reflects the effect on the stability of the protein that arises due to a change in the concentration of co-osmolyte(s) present, it provides a good measure of the efficacy of osmolytes in forcing the protein to fold [74,75,26]. m-Values can readily be obtained experimentally by fitting the respective folding/unfolding cooperative transitions to the linear extrapolation method (LEM) [88– 91]. From such fitting curves, one can also obtain the standard free-energy change in the presence of osmolyte [26]. We have calculated these apparent thermodynamic parameters of osmolyte-induced folding from the experimental data [48]. Since unfavorable interaction and simultaneous compatibility of osmolytes with the protein backbone accounts for the increased protein folding/stability, then altered strength of hydrophobic interactions could lead to non-specific effects such as protein aggregation, which could be detrimental to protein structure and functions in vivo [92]. However, some studies have shown that osmolyte has virtually no effect on the strength of hydrophobic interactions [92,93]. The neutrality of osmolyte towards hydrophobic interactions is manifested due in part to the lack of strong preferential binding or its depletion in the vicinity of hydrophobic solutes, suggesting that at high concentrations, osmolytes can be compatible with cellular components through its neutral approach for hydrophobic interactions [92,93]. Further, thermodynamic calculations indicate that the amphiphilic character of osmolytes may likely be responsible for their neutral behavior towards hydrophobic interactions [92]. However, this behavior does
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not provide an explanation for the increased stability of proteins in the presence of osmolytes.
Summary and perspectives It has been emphasized that the role of protein backbone is critical in determining thermodynamic stability of proteins in osmolyte solutions, and that the water-mediated interaction between protein backbone and organic osmolytes is unfavorable that makes the unfolded conformations of proteins less stable relative to folded states in the presence of specific osmolytes [53,94–97]. Therefore, designing small molecules that can provide unfavorable interactions with the protein backbone appears to be an excellent strategy towards protein structure stabilization. Since all proteins possess a backbone, irrespective of the composition of amino acid chains, therefore, stabilization of protein structures using osmolytes could be a critical step in preventing various critical proteins from misfolding or aggregating [6]. This may have far reaching consequences in understanding and preventing several deleterious diseases that are caused by protein misfolding/aggregation [36– 38,41]. Since organic osmolytes are naturally occurring molecules, they can have potential therapeutic applications without fear of major toxic side effects [98]. It has been shown that oral administration of an osmolyte can significantly inhibit polyglutaminemediated protein aggregation in the transgenic mouse model of Huntington disease and subsequently increased life span [98]. This study further suggests that the beneficial effects of osmolyte are due mainly to stabilizing the partially unfolded Huntington protein containing elongated polyglutamine chain length [98]. Another study has demonstrated that osmolytes can be used to fold androgen receptor containing elongated polyglutamine chain length in its N-terminal domain that possesses a powerful transactivation domain, which is otherwise intrinsically disordered in solution [99,100]. It has been reported that this elongated polyglutamine chain length in the androgen receptor leads to formation of protein aggregates and is responsible for a neurodegenerative disease [99,100]. It has also been shown that these effects take place at the level of gene regulation [99,100]. Further, it has been hypothesized that elongated polyglutamine chain length in the androgen receptor interferes with the ability of androgen receptor to interact with certain critical coregulatory proteins, essential for gene regulation by the androgen receptor [101]. We have earlier reported that presence of osmolyte can significantly enhance the interaction of androgen receptor with such critical coregulatory proteins [47]. Therefore, it is logical to build upon the hypothesis that osmolytes can be used to maintain proper functioning structure of androgen receptor in particular and may be extended to other related transcription factors in general. The prospect of using osmolytes as a therapeutic tool for neurodegenerative diseases appears to be quite exciting because they are known to be generally safe. However, there are many steps between an exciting prospect and an actual, safe treatment. While the initial results are promising, more studies need to be performed to demonstrate their effectiveness. This protein aggregation/misfolding process constitutes a hallmark of neurodegenerative pathologies, including Alzheimer’s, Huntington’s, and Parkinson’s diseases, and if osmolytes can provide a unifying mechanism of action, this may have far reaching consequences in developing better therapeutic tools for the management of such diseases. Such effects of osmolytes on protein folding pathways have become important to study. Under physiological conditions, the cellular compositions of osmolytes may vary significantly; therefore, different protein folding pathways utilized in cell may depend upon the cellular environment within it. Understanding the role of osmolytes in cell regulation will not only allow to predict the action of osmolytes on macromolecular interactions
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Review
Role of naturally occurring osmolytes in protein folding and stability Raj Kumar * Department of Basic Sciences, The Commonwealth Medical College, 501 Madison Avenue, Suite 205, Scranton, PA 18510, USA
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Article history: Received 29 August 2009 and in revised form 14 September 2009 Available online 19 September 2009 Keywords: Osmolyte Protein folding Protein structure Cooperativity Protein backbone Amino acid side chain
a b s t r a c t Osmolytes are typically accumulated in the intracellular environment at relatively high concentrations when cells/tissues are subjected to stress conditions. Osmolytes are common in a variety of organisms, including microorganisms, plants, and animals. They enhance thermodynamic stability of proteins by providing natively folded conformations without perturbing other cellular processes. By burying the backbone into the core of folded proteins, osmolytes can provide significant stability to proteins. Two properties of osmolytes are particularly important: (i) their ability to impart increased thermodynamic stability to folded proteins; and (ii) their compatibility in the intracellular environment at high concentrations. Under physiological conditions, the cellular compositions of osmolytes may vary significantly. This may lead to different protein folding pathways utilized in cells depending upon the intracellular environment. Proper understanding of the role of osmolytes in cell regulation should allow predicting the action of osmolytes on macromolecular interactions in stressed and crowded environments typical of cellular conditions. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Proteins need to maintain their natively folded structures for proper functions under physiological conditions [1]. However, most proteins are sensitive to changes in cellular and environmental conditions including temperature, pressure, and the presence of salts and other solutes [2–4]. Therefore, under physiological conditions, proteins require to counter-balance any significant perturbations in these thermodynamic conditions to avoid significant changes in their secondary and tertiary structures [5,6]. Protein failing to adapt to such conditions may result in a partial or complete loss of their functional activity [2–4]. In order to adapt to such perturbations due to extreme conditions, nature has created certain mechanisms such as accumulation of small organic solutes, also known as ‘‘osmolytes” [5–7]. Osmolytes are naturally occurring small organic molecules often referred to as ‘‘chemical chaperones”, and are typically accumulated in the intracellular environment at relatively high concentrations that can increase thermodynamic stability of folded proteins without perturbing other cellular processes [4,7–10]. In many cases, the environmental stress due to harsh denaturation conditions, threatens the stability of intracellular macromolecules [2–4,11,12]. Therefore, many osmolytes have been selected for their ability to stabilize cell components such as proteins [13]. These naturally occurring compounds exert a force to fold proteins that are highly unstructured in an aqueous environment [14,10,15]. It has been demonstrated * Fax: +1 570 504 9660. E-mail address: [email protected]. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.09.007
that osmolytes can cause proteins to fold cooperatively into native-like, functional species [16–19]. It has been shown that the protein backbone is effectively osmophobic, and osmolytes can provide significant stability to protein by hiding the backbone into the core of folded proteins [20–22]. The ability of osmolyte to increase the driving force for protein folding is due to a solvophobic effect on the peptide backbone exposed in the unfolded state [23–25]. Another feature of these molecules is that they make the unfolded state of protein in osmolyte solution very unfavorable relative to the folded state, without changing the rules of protein folding that occur in dilute solutions [26,27]. It is generally acknowledged that the functions carried out by proteins depend upon their structures [1]. It is assumed, based on the pioneering work of Anfinsen and coworkers that the primary sequence of a protein contains all the information required for it to fold properly [28]. However, it is not yet possible to predict with certainty the folded form of a protein from its primary sequence [1,6]. Since the peptide chains of all proteins have an identical composition, how do the side chains dictate overall folding? Also because of an astronomically large number of possible polypeptide chain conformations, how does a given sequence find its specific native structure in a finite time? There are suggestions that once proteins are synthesized, the highly disordered unfolded state pass through well-defined partially structured transition states, to fully folded forms (Fig. 1) [29]. After synthesis on the ribosome, protein is assumed to fold in the endoplasmic reticulum, aided by molecular chaperones that deter aggregation of incompletely folded species [29]. The correctly folded protein is secreted from the cell and functions normally in its extracellular environment
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pared with the buffer [52]. On the other hand, same is not true for another protease, chymotrypsin [52], suggesting that effects of osmolytes on the protein folding/stability are unique in nature and could vary [26]. Thus osmolytes may be an answer to such problems associated with protein stability/folding [7,26]. In this review article, we have summarized an up dated knowledge of presence of various organic osmolytes in nature, and their role in maintaining/inducing folding and/or stability of macromolecules that could lead to better management of various cellular and physiological functions of proteins.
Osmolytes in nature
Fig. 1. Schematic diagram of some of the states accessible to a polypeptide chain following its biosynthesis. There are suggestions that once proteins are synthesized, the highly disordered unfolded/unstructured state pass through well-defined partially structured/folded transition states (intermediate stage), to fully native folded and functionally active conformation (final stage). After synthesis on the ribosome, protein is assumed to fold in the endoplasmic reticulum, aided by molecular chaperones that deter aggregation of incompletely folded species. The correctly folded protein is secreted from the cell and functions normally in its extracellular environment. Under certain conditions protein unfolds, at least partially, and becomes prone to aggregation. This can result in the formation of fibrils or amyloids and other aggregates that accumulate in tissue/cell. It is likely that small aggregates as well as the highly organized fibrils and plaques, can give rise to pathological conditions, at least in some cases. Naturally occurring osmolytes could facilitate the intermediate reactions by shifting it to the final natively folded conformation, thereby making them less susceptible to aggregation. U, unfolded state; I, partially structured/folded intermediates; N, globular native form (based on Ref. [29]).
[29]. Intracellular proteins are also aided by several chaperone proteins to maintain their proper structure under physiological conditions [30,31]. Under certain conditions, protein unfolds, at least partially, and becomes prone to aggregation [32–35]. This can result in the formation of fibrils and other possible aggregates that accumulate in tissue [36–41]. It is likely that small aggregates as well as the highly organized fibrils and plaques, can give rise to pathological conditions, at least in some cases [42,43]. The question then is how the ensembles of unfolded or partially folded proteins are in such a small population to avoid aggregation in the biological environment. The answer to this possibly lies with the cooperative nature of protein folding process [16–19]. In the two-state model of protein folding, the partially folded intermediate states are small [44]. Cooperativity of protein folding process has long been documented in well-defined native proteins capable of carrying their functions [16–19]. It seems that cooperative nature of protein folding could be equally crucial for avoiding protein/ peptide aggregation [16–19]. In fact, several studies, including ours have shown that in the presence of certain osmolytes, long stretches of unstructured or intrinsically disordered regions/domains of proteins can be folded in a cooperative manner, and the resulting conformations are native-like with significant functional activities [22,44–50]. Many of these intrinsically disordered regions are found in the activation domains of various transcription factors, which are involved in the gene regulation [51]. There are some reports showing that even a relatively folded protein with an active globular structure can also adopt a more functionally active conformation in the presence of osmolyte [52]. We have earlier shown that in the presence of osmolyte, trimethylamine-Noxide (TMAO)1, trypsin, a proteolytic enzyme can adopt a conformation that facilitates its enzyme activity by several fold when com1 Abbreviations used: TMAO, trimethylamine-N-oxide; LEM, linear extrapolation method.
As discussed above, organic osmolytes are used widely in nature to protect cellular proteins against harsh conditions such as the effects of dehydrations, other hypertonic states, or the buildup of potentially denaturing metabolites [4,53–61]. Hyper-osmotic conditions set off a response in most cell types that turns on the synthesis of protective organic osmolytes [7]. There are three major chemical classes of protecting osmolytes, and while they all have varying abilities to fold/stabilize proteins, they also have different physical and chemical properties that may alter the function of a second osmolyte present in cells [4]. A list of some common categories of the osmolytes is shown in Fig. 2. The selection of osmolytes depends on the duration of the osmotic stress and the availability of substrates and osmolytes in the surroundings [7,26]. In deep sea invertebrates, high levels of several osmolytes are present [57]. To balance the high osmolality of sea water and to counter-balance high concentrations of urea, these animals accumulate organic osmolytes in their tissues and extracellular fluid, particularly in the elasmobranchs [4]. Of particular interests are the methylamines that accumulate with urea to counteract its denaturing effects [26]. Since the most important determinant of the difference between protein denaturant (e.g. urea) and osmolytes (e.g. methylamines) is their opposite effects on the configuration of protein backbone, therefore, at a particular ratio of methylamines to urea (generally 2:1), the denaturing effects of urea are counteracted [62]. Yeast and fungi are known to generate and/or accumulate different osmolytes from the environment [63,64]. Glycerol is accumulated in response to hyper-tonicity by increasing its production and reducing its efflux [63,64]. Besides several others, high levels of glycerol are employed as an osmolyte by Saccharomyces cerevisiae [63,64]. Several different organic osmolytes are reported to be accumulated in bacteria (particularly Escherichia coli and Salmonella typhimurium) in response to hypertonicity [65]. When growing in the absence of exogenous osmolytes, E. coli cells increase the synthesis of certain osmolytes e.g. trehalose [65]. Organic osmolytes generally are accumulated in plants by increasing the rate at which they are synthesized [66]. Plants subject to salt stress accumulate organic osmolytes in the cytoplasm of their cells [66]. The increased accumulation of osmolytes in leaves of stressed plants is partly regulated through changes in the gene expression of their biosynthetic enzymes [67]. However, the mechanism of action for these changes has not been well understood. The osmolality of mammalian blood is normally kept constant by a combination of thirst and varying urinary concentrations [19]. Therefore, most mammalian cells are not normally exposed to the extreme osmolalities experienced by several other kind of non-mammalian cells [4,19]. However, certain mammalian cells contain considerable concentrations of organic osmolytes [4,64]. Renal medullary cells are known to contain the highest levels of organic osmolytes as a consequence of being exposed to extremely high concentrations of salt and urea [4,19,64]. The mechanisms of accumulation and their regulation in response to high NaCl
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Fig. 2. Diagram showing two examples each in three major categories of osmolytes. There are a number of well-known naturally occurring osmolytes which fall into three chemical classes: methylamines, such as TMAO, Choline-O-sulphate, and sarcosine: polyols such as sorbitol, glycerol, sucrose, and trehalose; and certain amino acids, such as glycine, taurine, proline, and betaine. Each class interacts with both the peptide backbone and to varying extent, also with amino acid side chains. The balance of these effects determines the potency of the osmolyte to promote protein folding and solubility. Thermodynamic calculations and experimental results support the view that the powerful solvophobic effects of osmolytes on the peptide backbone dominate, such that the relative Gibbs free energy of the unfolded state is less favorable than that of the folded state.
and urea have been most thoroughly studied in renal medullary cells [64]. Aside from renal cells, the normal levels of organic osmolytes in specific cell types have neither been surveyed intensively nor have been examined in many tissues under pathophysiological conditions, but it has been established that in many cells/ tissues it may well reach in a very high concentration range [64]. It has been calculated that osmolyte concentrations in whole tissues may often reach in the range of 300–400 mmols/kg of cell water, meaning that in certain compartments, they are almost surely much higher [7]. Desert mouse kidney is one extreme case where the concentration of an osmolyte in this animal occurs there at 2.5 M, allowing retention of structure and function of essential proteins [4,7]. This is because in this case the urea concentration may reach up to 5 M [4,7]. Trimethylamine-N-oxide (TMAO), an osmolyte extensively studied for protein folding, is natural solute with high concentrations in the shark, where it exists to prevent the denaturant effect of the high urea levels in those animals [4,7,26]. In fact, it is common to find more than one intracellular organic osmolyte in cells [4,7]. Human kidney, for example, contains several osmolytes, including glycine betaine, sorbitol, inositol and taurine [4,7]. The presence of several osmolytes inside cells raises a number of questions about their roles in protecting intracellular macromolecules against the environmental stresses. The most immediate question is whether these osmolytes work individually in an independent manner and/or in conjunction with each other. If osmolytes produce their effects in combinations; are these effects synergistic in their ability to protect cellular components against potentially denaturation conditions that cells may encounter. Even in the same cell, there could be many different osmolytes present [4,7]. However, their concentrations and compositions may vary in different cell types [64]. Since the protection provided by an osmolyte does not depend on specific chemical interactions with the macromolecules, in principle any of the osmolytes should be capable of replacing each other, depending upon either endogenous or exogenous availability of particular osmolyte(s) [64]. There are some reports to support this idea [68–72]. For example, when renal cells in culture are maintained at a constantly high salt concentration, an induced change in an organic osmolyte is reciprocated by an opposite change in the amount of another one with similar protective effects [73]. This raises the question about the natural selection criteria for osmolytes in cells. Why do cells need so many osmolytes, and why are there so many classes of osmo-
lytes? Given their interchangeable protective effects, why should one osmolyte be preferred over another? The explanation may be that even though the protective effects of osmolytes are non-specific, an osmolyte or the mechanisms that regulate it may have specific effects. Bolen’s laboratory has recently shown that the effects of differing osmolytes are additive, so that a cell may contain even higher summative concentrations of these protein pro-folding molecules [74,75]. A hypothetical model of such actions of osmolytes on the folding of an unstructured protein is shown in Fig. 3. Of course, membrane-limited intracellular compartmentalization in the cell and the intracellular environment may also make for quite high concentrations in specific loci or compartments [74,75]. Therefore, it is logical that cells do not routinely express extremely high levels of osmolytes [26]. Several classes of well-known organic osmolytes are present in mammalian cells, where they function to protect proteins from denaturation under certain conditions [1–7]. Since, many cellular proteins function through interaction with other binding protein partners to form large assembly of protein complexes that must have enough flexible conformation(s) to include or exclude binding partners in response to specific cellular functions; it must not have too rigid structure [48,51]. Therefore, maintenance of excessive amounts of folded proteins would interfere with release of bound protein partners that require a structural relaxation, with the loss of conformation required for proteolysis and turnover and maintenance of normal osmotic, salt-based transmembrane gradients [20]. Therefore, it is possible that specific cells utilize specific osmolyte(s) to encourage optimal functioning conformation(s) of certain proteins [7]. This may in fact be true for several transcription factors, which possess intrinsically disordered activation domains, and have cell-specific effects in gene regulation [51]. Based on our findings with the intrinsically disordered activation domain of steroid receptors [51], it can be hypothesized that cell-specific effects of signals passed by steroids through their receptor to regulate the expression of specific target gene may be dependent in part to the presence of specific sets and concentrations of cellular milieu.
Osmolyte-induced protein folding Protein folding process is reversible in nature, and the equilibrium between unfolded and natively folded does not involve cova-
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Fig. 3. A model showing osmolyte-induced characteristics in thermodynamic terms of solvophobic effects. Osmolytes interact unfavorably with the unfolded or unstructured states, resulting in preferential depletion of osmolyte proximate to the protein surface, thereby forcing protein to adopt folded conformation(s). Protein is shown with green color. In the center, protein is intrinsically disordered or unstructured, and is surrounded by only water molecules (small blue filled circle). O1, O2, O3, or O4 (shown by large filled circle, red, green, blue, and purple, respectively) represent the presence of various osmolytes (Ox). In the presence of each osmolyte, due to sequestering of water molecules by osmolytes, protein adopts folded conformation, which may have similar or different overall fold depending upon the nature of osmolyte and protein. The final natively folded conformation of protein may be determined by the sum of these osmolytes. Effects of these osmolytes on the folding of protein may be additive in nature. Thus, the availability of number of osmolytes in a particular cell may decide the concentration of osmolytes required to fold protein into a functionally active conformation. The final outcome may also be determined by the nature of other interacting macromolecules and small ligands (if any).
lent bonds during inter-conversion between unfolded and natively folded conformations [20]. Therefore, a thermodynamic process of protein folding can be framed in terms of solvent interactions with the unfolded and native states [20]. Osmolytes push the folding equilibrium toward natively folded conformations, whereas denaturing solutes such as urea push the equilibrium toward unfolded conformations [20]. The protecting osmolytes raise the free-energy of the unfolded state, favoring the folded population, whereas denaturing solutes lower the free-energy of the unfolded state, favoring the unfolded population [20]. Accordingly, osmolytes interact unfavorably with the unfolded state, resulting in a preferential depletion of osmolyte proximate to the protein surface [20]. A model for such osmolyte-induced characteristics has been shown in thermodynamic terms (Fig. 4). However, there are certain limits to this phenomenon, and thus it cannot be termed as a universal theory that can account for the mechanism by which osmolytes interact with the protein to affect stability [20]. There are reports showing that the osmolytes predominantly affect the protein backbone, a common component to all polypeptide chains [26]. Though the magnitude of the energy of interactions with each peptide bond is very small, peptide bonds are by far the most numerous structural component of a protein. Consequently, the sum of such interactions can be quite large [20,75,26]. It is the balance between osmolyte-backbone interactions and amino acid side chain-solvent interactions that determines the outcome on protein folding [26]. Intrinsically disordered or unstructured regions do not of themselves contain sufficient hydrophobic amino acid side chains to fold spontaneously [51,76–80]. Thus, addition of an osmolyte tips the balance to a favorable negative free energy for folding [26]. One interesting aspect of osmolyte-induced protein folding is a very weak protein:osmolyte interaction, and therefore, the free-en-
Fig. 4. A schematic diagram showing protein stabilization brought about by the action of osmolyte. Osmolytes act by increasing DG between the natively folded (Nbuffer) and unfolded ensembles. DG1 is the unfolding Gibbs energy difference between native and unfolded (Ubuffer) protein in aqueous buffer solution, and DG3 is the Gibbs energy change for the same reaction in the presence of an osmolyte. Transfer of Nbuffer or Ubuffer from aqueous buffer to osmolyte solution (Nosmolyte and Uosmolyte, respectively) raises the DG2 of the unfolded ensemble much more than it does for the natively folded ensemble (DG4), resulting in a greater stability of the protein in the osmolyte solution than in aqueous solution (based on Ref. [44,62]).
ergy effects are additive because osmolytes do not occupy any significant fraction of the backbone surface [20,26]. This essentially allows no competition for backbone binding sites [26]. The natural protein folding force derives primarily from the composition and distribution of amino acid side chains [28]. In unstructured proteins, the hydrophobic amino acids are insufficient, relative to the charged side chains, to cause spontaneous
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folding [81–85]. Addition of the solvophobic effect through addition of an osmolyte can tip the free-energy balance in the favor of folding, as the backbone collapses to avoid solvent with osmolyte [26]. The quantity of osmolyte(s) required depends on both its inherent solvophobic effects in the interaction with peptide backbone, and the free-energy balance provided by the sum of all backbone-osmolyte interactions and the sum of all amino acid side chain-solvent interactions [74,75,26]. Because protein backbone comprises the most numerous functional groups in proteins, osmolyte-induced conformations are driven by very strong forces, and evidence from the several systems studied thus far indicates native folded functional species result [51,26]. Given the essential roles of osmolytes in protecting cells, it is important to determine how effect of one osmolyte on protein stability is affected by the presence of other osmolyte(s). One of the primary roles of the thermodynamic parameters is to attain predictive understanding of the energetic changes, which are responsible for folding/unfolding of proteins at the level of amino acids [26]. Major forces, which are fundamental to our understanding of protein folding and stability, can be attributed to the transfer of free-energies that accompanies the transfer of amino acid side chains and peptide backbones from water to other solvents, and thereby measuring the hydrophobicity and hydrogen bonding of proteins [86–88]. Since, it is well established that the peptide backbone makes the dominant contribution to the free-energy change between the native and denatured states, these thermodynamic approaches have been extensively used to determine osmolyte-induced protein folding [26]. Because organic osmolytes within the cellular milieu stabilize intracellular proteins to protect cells against denaturation conditions, a direct link between cellular function and energetics of protein folding becomes a central issue of solvent–protein interactions in vivo under physiological conditions [86–88]. Auton and Bolen recently demonstrated that free-energies of side chain and backbone transfer from water to osmolytes can be achieved by predicting solventdependent cooperative protein folding/unfolding free-energy changes (in terms of m values) [87]. An experimental measure of the effect of an osmolyte on protein stability can be determined from the m-value, obtained from the slope of a plot of the native to denatured free-energy change as a function of osmolyte concentration [74,75,26]. Since the mvalue reflects the effect on the stability of the protein that arises due to a change in the concentration of co-osmolyte(s) present, it provides a good measure of the efficacy of osmolytes in forcing the protein to fold [74,75,26]. m-Values can readily be obtained experimentally by fitting the respective folding/unfolding cooperative transitions to the linear extrapolation method (LEM) [88– 91]. From such fitting curves, one can also obtain the standard free-energy change in the presence of osmolyte [26]. We have calculated these apparent thermodynamic parameters of osmolyte-induced folding from the experimental data [48]. Since unfavorable interaction and simultaneous compatibility of osmolytes with the protein backbone accounts for the increased protein folding/stability, then altered strength of hydrophobic interactions could lead to non-specific effects such as protein aggregation, which could be detrimental to protein structure and functions in vivo [92]. However, some studies have shown that osmolyte has virtually no effect on the strength of hydrophobic interactions [92,93]. The neutrality of osmolyte towards hydrophobic interactions is manifested due in part to the lack of strong preferential binding or its depletion in the vicinity of hydrophobic solutes, suggesting that at high concentrations, osmolytes can be compatible with cellular components through its neutral approach for hydrophobic interactions [92,93]. Further, thermodynamic calculations indicate that the amphiphilic character of osmolytes may likely be responsible for their neutral behavior towards hydrophobic interactions [92]. However, this behavior does
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not provide an explanation for the increased stability of proteins in the presence of osmolytes.
Summary and perspectives It has been emphasized that the role of protein backbone is critical in determining thermodynamic stability of proteins in osmolyte solutions, and that the water-mediated interaction between protein backbone and organic osmolytes is unfavorable that makes the unfolded conformations of proteins less stable relative to folded states in the presence of specific osmolytes [53,94–97]. Therefore, designing small molecules that can provide unfavorable interactions with the protein backbone appears to be an excellent strategy towards protein structure stabilization. Since all proteins possess a backbone, irrespective of the composition of amino acid chains, therefore, stabilization of protein structures using osmolytes could be a critical step in preventing various critical proteins from misfolding or aggregating [6]. This may have far reaching consequences in understanding and preventing several deleterious diseases that are caused by protein misfolding/aggregation [36– 38,41]. Since organic osmolytes are naturally occurring molecules, they can have potential therapeutic applications without fear of major toxic side effects [98]. It has been shown that oral administration of an osmolyte can significantly inhibit polyglutaminemediated protein aggregation in the transgenic mouse model of Huntington disease and subsequently increased life span [98]. This study further suggests that the beneficial effects of osmolyte are due mainly to stabilizing the partially unfolded Huntington protein containing elongated polyglutamine chain length [98]. Another study has demonstrated that osmolytes can be used to fold androgen receptor containing elongated polyglutamine chain length in its N-terminal domain that possesses a powerful transactivation domain, which is otherwise intrinsically disordered in solution [99,100]. It has been reported that this elongated polyglutamine chain length in the androgen receptor leads to formation of protein aggregates and is responsible for a neurodegenerative disease [99,100]. It has also been shown that these effects take place at the level of gene regulation [99,100]. Further, it has been hypothesized that elongated polyglutamine chain length in the androgen receptor interferes with the ability of androgen receptor to interact with certain critical coregulatory proteins, essential for gene regulation by the androgen receptor [101]. We have earlier reported that presence of osmolyte can significantly enhance the interaction of androgen receptor with such critical coregulatory proteins [47]. Therefore, it is logical to build upon the hypothesis that osmolytes can be used to maintain proper functioning structure of androgen receptor in particular and may be extended to other related transcription factors in general. The prospect of using osmolytes as a therapeutic tool for neurodegenerative diseases appears to be quite exciting because they are known to be generally safe. However, there are many steps between an exciting prospect and an actual, safe treatment. While the initial results are promising, more studies need to be performed to demonstrate their effectiveness. This protein aggregation/misfolding process constitutes a hallmark of neurodegenerative pathologies, including Alzheimer’s, Huntington’s, and Parkinson’s diseases, and if osmolytes can provide a unifying mechanism of action, this may have far reaching consequences in developing better therapeutic tools for the management of such diseases. Such effects of osmolytes on protein folding pathways have become important to study. Under physiological conditions, the cellular compositions of osmolytes may vary significantly; therefore, different protein folding pathways utilized in cell may depend upon the cellular environment within it. Understanding the role of osmolytes in cell regulation will not only allow to predict the action of osmolytes on macromolecular interactions
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