Osmolytes Promote Protein Folding

Chaperoning Your
Proteins to Better Health

Organic compounds called osmolytes protect
our proteins from misfolding, and our cells from stress
By Will Block

uppose you had taken a class in advanced gift packaging, including the creation of complex bows, full of intricately crafted twists and loops. You’re practicing your newfound skill on a birthday gift for a loved one. Unbeknownst to you, your kitty has been crouching in a favorite hiding place, watching intently, tail twitching fitfully, as the beautiful creation slowly takes shape. The cat is thinking . . . well, who knows what cats think? But we know what they do, don’t we? Perhaps the gift needs a chaperone (the family dog?) to help keep it intact when you leave the room.

In every cell of your body, chemical compounds called chaperones help protect the intricately folded molecular structures of your proteins from coming unraveled at the claws of cellular “kitties” whose capacity for mischief is boundless. The kitties are certain compounds that act as protein denaturants, meaning that they cause the protein to unravel from its correct three-dimensional configuration, called the native state. When the native state is lost, so is the protein’s function, and there can be physiological hell to pay—more on that later.

A Sharp Claw or a Soft Paw?

The chemical kitties’ “claws” are the atomic force fields they possess. The particular pattern of force fields existing at the surface of a molecule is like a fingerprint, unique to every chemical compound, from the smallest to the largest—although many molecules have certain features in common, giving them similar chemical characteristics. Whether a molecule’s pattern of force fields tears at that of another molecule like a sharp claw or caresses it like a soft paw depends on the details of the patterns in question (it’s all in the quantum mechanics). Some interactions are destined to be hostile, in a chemical sense, whereas others are destined to be friendly.

Understanding the details of these interactions—why they occur, how they occur, what the results will be, and how these results can be modified by changing the process conditions—is the essence of the science of chemistry. And since chemistry is the foundation science of biology (just as physics is the foundation science of chemistry), life’s processes can be understood only in terms of the chemical interactions that underlie them. That’s what biochemistry and molecular biology are all about.

Protein Misfolding—A Huge Problem

Which brings us back to proteins, whose intricate structures, full of weird twists and loops, make them the most complex of all molecules. In last month’s issue, we published an article ( “Youthful Aging Depends on Proper Protein Folding”) in which we described the basics of protein structure. The central feature is the tertiary structure, achieved via the folding of the amino acid chain to its ultimate, optimal, compact, three-dimensional configuration—the native state. (Having read that article will be a help in understanding this one.)

The problem with proteins is their tendency, in some circumstances, to become misfolded, i.e., folded incompletely or incorrectly, such that their correct function—which derives exclusively from their correct molecular structure—is degraded or lost. It has been estimated that about half of all human diseases are caused by protein folding defects, so this phenomenon is clearly of huge importance.1 Protein misfolding is also thought to be a factor in the aging process, making it that much more important that we understand misfolding and devise ways to counteract it.

Because misfolding alters the conformation of a protein, disorders that result from this defect are called conformational disorders (or, sometimes, folding disorders). They tend to be age-related, and they can result in cell toxicity, functional impairment, and even death. But why do proteins become misfolded? There are two causes: genetics and stress. Let’s take them in that order.

Genetic Mutations Can Cause Misfolding

When a mutation occurs in a gene that encodes the amino acid sequence of a protein, that sequence may be altered, often in the form of a substitution of one amino acid for another. (All of our proteins are composed of 20 different amino acids, in polymeric chains containing dozens, hundreds, or thousands of them.) Depending on the nature of the alteration and where it occurs in the chain, the protein structure and function that result when the chain folds up (i.e., when it misfolds compared with the correct configuration), will be incorrect in ways that can range from trivial to fatal.*

*An example of the latter is sickle cell anemia, which is caused by the substitution of one valine residue for one glutamic acid residue in the amino acid chain of the hemoglobin molecule (amino acids in a protein chain are called residues). This was discovered by Linus Pauling in 1949—it was the first time that any human disease was explained at the purely molecular level.

Often the chain may not even get the chance to fold up fully, because biochemical quality control mechanisms will identify the structure as alien to the cell and will engineer its destruction. This is accomplished by efficient molecular “garbage disposals” called proteasomes, which are themselves large protein complexes (there’s an irony for you). [For more on this grisly process, see the sidebar “Demolition Derby with Proteasomes” in the article “Resveratrol Fights Brain Plaque” (November 2005).]

Cellular Stress Can Cause Misfolding

The other cause of protein misfolding—including the misfolding of proteins that were normal to begin with—is cellular stress of various kinds. And it doesn’t take much stress to begin the process. In terms of chemical energetics (thermodynamics), the difference in stability between the native state of most proteins and their unfolded state is very small.

This means that most proteins are highly vulnerable to degradation (think cotton candy vs. steel wool), ranging from a minor unraveling of the chain to full denaturation (a complete unraveling). Such damage, including a misfolding of the chain to an incorrect tertiary structure, can be caused by excessive heat (e.g., when we have a fever), excessive cold (hypothermia), ionizing radiation, or a variety of chemical stresses (such as the presence of free radicals) and mechanical stresses (such as protein overcrowding caused by cell shrinkage).

Protein Aggregation Can Cause Physiological Havoc

One of the worst consequences of protein misfolding is aggregation, a clumping together of defective (or not yet folded) proteins due to their enhanced surface reactivity or due simply to protein overcrowding (which can also cause normal proteins to aggregate). Protein aggregation can lead to degradation of cellular function and overt disease. The most common example is amyloidosis, the collective name for diseases characterized by the accumulation of amyloid (a group of chemically diverse proteins) as harmful deposits in various organs or tissues of the body.

If this sounds horribly familiar, it’s because you know about the worst amyloid of all, amyloid-beta, which forms the brain-destroying plaques in Alzheimer’s disease. (The importance of protein misfolding here is underscored by the existence of a medical journal called Amyloid: The Journal of Protein Folding Disorders.)

The other fate that awaits misfolded proteins is destruction by the cellular quality control mechanism (aka cellular housekeeping) mentioned above. In this case, at least, the offending proteins are not allowed to clog up the cellular machinery of life, but their disposal can create a deficit in the cell’s normal complement of the protein in question, and hence a degradation of cellular function.

All Hail Our Chaperones!

Fortunately, as mentioned above, evolution has given us chaperones—chemical compounds that help protect our proteins from misfolding, via complex mechanisms whose details are still being, uh, unraveled by scientists. One type, called molecular chaperones, is composed of specialized proteins (another irony) that evolved to be tougher than most other proteins and to fulfill this protective function—sort of like rottweilers guarding lapdogs. Because they’re activated under conditions of excessive heat as well as other forms of cellular stress, these compounds are also known as heat-shock proteins. [For more on molecular chaperones and protein folding, see the article “Clear Eyes with N-Acetylcarnosine” (December 2006).]

The other main type of natural chaperone, called chemical chaperones, consists of various small organic molecules—tiny and very simple compared with proteins—whose chemical properties (which are determined by their atomic force fields, remember?) allow them to mimic the actions of molecular chaperones: they can influence unfolded proteins to fold properly and misfolded proteins to refold properly.* Believe it or not. Without them, life as we know it would be impossible.

*There is a third category of chaperones—pharmacological chaperones—consisting of synthetic compounds designed by chemists to target specific proteins whose misfolding is implicated in a given disease, such as cancer or cystic fibrosis.2 Even pain relief may be associated with the refolding of a particular protein; the pharmacological chaperone naltrexone, an antinarcotic drug, is believed to accomplish this.

The Two Kinds of Chemical Chaperones

Now, there are two main kinds of chemical chaperones.2 One kind consists of certain compounds whose common denominator is that they’re hydrophobic, or “water-hating,” i.e., they’re repelled by water (or they repel water—same thing) and will therefore not dissolve well in it. The opposite of hydrophobic is hydrophilic, or “water-loving,” a characteristic of water-soluble compounds. These phenomena play key roles in all aspects of cellular biochemistry (as does the molecular structure of liquid water, which is highly complex and variable). Hydrophobic interactions, in particular, are a profoundly important factor in the mechanism of protein folding (this was elucidated in 1959 by the American physical chemist Walter Kauzmann).

The other kind of chemical chaperone is called osmolytes, and it’s the kind we’re most interested in here. Osmolytes are small organic compounds that play a key role in osmoregulation, which is the regulation of the water content and solute concentrations in our cells. As you may have guessed, this has something to do—a lot to do, actually—with osmosis, a term you’re surely familiar with. (But do you know what osmosis really is, and how it works? If not, the sidebar will help.)

The Beauty of Osmosis

Osmosis occurs throughout your body all the time. It’s one of life’s fundamental processes and is the key to kidney function. Osmosis is easy to define—it’s the passage of a solvent through a semipermeable membrane separating two solutions of different concentrations. A semipermeable membrane is one through which the molecules of the solvent (usually water) can pass, but not the molecules or ions of most solutes (e.g., sugar or salt).

The cell walls in our bodies (indeed, in all living organisms) act as semipermeable membranes that allow the passage not only of water molecules but also of some solutes. They are the sites of very complicated forms of osmoregulation governed by chemical, neural, and hormonal stimuli (more on that below).

In basic osmosis, the solvent molecules (water, typically) pass spontaneously from the side with the weaker (less concentrated) solution to the side with the stronger (more concentrated) solution, until equilibrium is reached, i.e., until the concentrations are equal. (“Spontaneously” means that the process occurs with no external energy input.) In other words, the tendency of osmosis is to dilute the stronger solution; this means that the weaker solution becomes more concentrated as its water molecules pass to the other side.


(click on thumbnail
for full sized image)

For a dramatic example of osmosis, take a red blood cell. If you put the cell in pure water, it will immediately swell and burst, because water enters the cell, trying to dilute its contents. Conversely, if you put the cell in seawater, whose 3.5% salt concentration is much higher than that of the cell, it will shrivel and die, because water leaves the cell, trying to dilute the seawater. Finally, if you put the cell in physiologic saline solution (0.9% salt), nothing will happen, because that solution is just right—a perfect match, osmotically speaking. (That’s why using saline solution is so important when fluids are administered intravenously.)

It’s impossible to understand why osmosis works as it does except in terms of chemical thermodynamics, a major branch of physical chemistry. Suffice it to say that the driving force behind osmosis is a well-defined thermodynamic quantity called the Gibbs free energy, which is, in fact, the driving force for all spontaneous physicochemical processes (including, of course, protein folding) and hence for all spontaneous physiological processes.

Josiah Willard Gibbs
It was named for the mathematical physicist Josiah Willard Gibbs, who proposed it in an epochal series of papers on thermodynamics published in 1876–1878. Gibbs, a professor at Yale University, was the greatest American scientist of the nineteenth century, and one of the greatest scientists who ever lived.

It’s easy to see why precise osmoregulation is critically important to life. Living things are, of course, immensely complex in their chemistry, and the semipermeability of cell walls is highly selective and variable, depending on the organ or tissue in question and on the prevailing conditions. Natural osmoregulatory mechanisms are too varied and complex to discuss here, but we can give one oversimplified example.

A common form of cellular stress is osmotic stress, usually caused by solute concentrations in the extracellular fluid becoming higher than those inside the cells. When this occurs, the tendency of osmosis is to cause water to leave the cells so as to dilute the extracellular fluid. That, however, would cause the cells to shrink, causing a host of problems. One of these is an increased tendency for proteins to aggregate, owing to their closer proximity to each other inside the cell. Uh-oh!

Nature has devised an ingenious way around this problem, in the form of osmolytes, many of which are maintained at high concentrations in our cells.1 Under the type of osmotic stress mentioned above, the cellular osmolyte concentration is increased via the uptake of even more osmolyte molecules (via osmosis) from the extracellular fluid or via the synthesis of more of them inside the cell. This reduces the tendency for water molecules to leave the cell, thus helping to maintain normal cell volume, and it increases the viscosity of the cell’s contents. Both of these factors help to inhibit protein aggregation.

In this way, the fluctuating osmolyte concentrations act as a buffer against the potential damage caused by osmotic stress. The evolution of life on earth has endowed every organism with some version of this beautiful regulatory mechanism.


  1. Burg MB, Ferraris JD. Intracellular organic osmolytes: function and regulation. J Biol Chem 2008;283(12):7309-13.

Osmolytes Are Natural Protein Protectors

Natural selection has created a system of osmolytes that do double duty in our cells: in addition to the osmoregulation for which they’re named, many of them serve as chemical chaperones in the sense described above. Remarkably, there is evidence suggesting that some osmolytes, at least, may also serve as activators or regulators of molecular chaperones, indicating a degree of cooperativity in these cellular protein-protective networks.3

Some examples of osmolytes (most of which are found naturally in our tissues) are: the amine compound trimethylamine N-oxide (TMAO); the amino acids proline, glycine, and beta-alanine; the amino acid derivatives betaine (trimethylglycine), creatine, and taurine; the polyhydric alcohols inositol and glycerol; the sugars sucrose and trehalose; the versatile organic solvent dimethylsulfoxide (DMSO); and the nitrogenous waste product urea and its derivative guanidine. Some of these compounds may have therapeutic potential in humans, as we will see below.

With regard to osmoregulation, all osmolytes are beneficial by definition. With regard to protein folding, however, it’s a mixed bag: most osmolytes are beneficial (i.e., they act as chemical chaperones and are thus called protective osmolytes), but a few are detrimental, because they act as protein denaturants. The most notorious denaturing osmolyte (a clawing kitty, to invoke our opening imagery) is urea, a very simple molecule that is a waste product of nitrogen metabolism in all our cells.* An important task of the protective osmolytes is to shield our proteins from attack by urea, guanidine, and other denaturants.

*Urea (which, by the way, is an important agricultural and industrial chemical) occupies an honored place in the history of science: it was the first organic compound to be synthesized solely from inorganic precursors. This feat was achieved by the German chemist Friedrich Wöhler in 1828. It laid the groundwork for organic chemistry and was the first nail in the coffin of vitalism, the long-held belief that the molecules of living things had in them a unique, mysterious “life force.” A century later, in 1926, the enzyme urease, which decomposes urea to ammonia and carbon dioxide, became the first enzyme to be crystallized in the laboratory and the first to be shown incontrovertibly to be a protein (we now know that all enzymes are proteins). This was accomplished by the American biochemist James Sumner, who won the Nobel Prize in chemistry for it.

Osmolytes Do It with Hydrogen Bonds

Schematic diagram of the human muscle protein myoglobin, which contains 153 amino acids. The coils represent hydrogen-bonded α-helical segments of the amino acid chain.
How do osmolytes affect protein folding? Do you care? Scientists do, of course, and recent investigations have shed much new light on this intriguing question.4,5,6 The answer lies mainly in subtle interactions between the osmolytes and the protein backbone, mediated by the water molecules in which they’re all immersed. These interactions occur mainly via the transient making and breaking of a type of chemical bond called a hydrogen bond, which is very different from—and much weaker than—conventional chemical bonds (covalent and ionic).

The structure of liquid water depends critically on hydrogen bonds, as do the structures of proteins and nucleic acids, whose unique configurations could not exist without them. Hydrogen bonds also occur in many other aspects of biochemistry, and of chemistry in general (both organic and inorganic). Our understanding of them is due mainly to Linus Pauling. In his monumental book The Nature of the Chemical Bond and the Structure of Molecules and Crystals (which was published in 1939 and is the most often-cited science book in human history), he wrote,7

It has been recognized that hydrogen bonds restrain protein molecules to their native configurations, and I believe that, as the methods of structural chemistry are further applied to physiological problems, it will be found that the significance of the hydrogen bond for physiology is greater than that of any other single structural feature.

All that we have learned about physiology since then has borne his prediction out. Unfortunately, a discussion of hydrogen bonds is beyond the scope of this article. Suffice it to say, for our present purposes, that they are the key to understanding the ways in which osmolytes influence the protein folding process. This is true both of protective osmolytes (protein folders) and of denaturing osmolytes (protein unfolders), whose weak hydrogen-bonding interactions with the protein backbone are similar in some respects but different in others—different enough to account for their opposite effects.

Whither the Research?

To date, almost all research on the protein-protective role of osmolytes has focused on discovering how a given osmolyte works on a given protein by affecting the competition between folding and aggregation. Most of the studies have been conducted in the laboratory with chemical systems or with bacteria, such as Escherichia coli, but some have used cell cultures from higher organisms, such as rodents and even humans.

Such research has great academic value and also great value for the biotechnology and pharmaceutical industries, whose research and development are plagued by problems associated with protein misfolding and aggregation. The main objective there is to discover how to treat or prevent diseases with chemical chaperones or, better yet from the industries’ point of view, with pharmacological chaperones (because that’s where the money is).

It is hoped, of course, that research with natural osmolytes will prove to have therapeutic value for humans who are afflicted with conformational disorders or who are simply getting older. Although there is not yet any direct evidence that supplementing with natural osmolytes will have therapeutic or preventive value in humans, it’s an intriguing possibility in the view of some researchers.

Proline Is a Good Prospect

Of particular interest to some is the amino acid proline, one of the 20 amino acids of which human proteins are composed. Proline is known to act as an osmolyte, in the osmoregulatory sense, in bacteria, algae, fungi, plants, and marine invertebrates (but not, apparently, in mammals).8

Laboratory studies have also shown proline to be a protective osmolyte with regard to protein structure. From a chemical point of view, proline is interesting to scientists because of certain features of its molecular structure that make it unique among the nutritional amino acids. These features have a strong effect on proline’s properties when it’s incorporated in the amino acid chain of a protein, and they also affect its behavior as an osmolyte.

Normal red blood cell (left) and sickled cell (right). The misfolding of the mutant hemoglobin molecules (about 250 million per cell) in sickle cell anemia causes the cell to become misshapen, with dire results.
In one laboratory study with artificially modified proteins, it was found that proline effectively blocked or reversed protein aggregation in its early stages; in later stages, proline blocked further aggregation but did not reverse the damage already done.9 Significantly, these effects were paralleled by those observed in similar experiments (in the same study) with E. coli, a living organism.

Combinations of Osmolytes May Be Beneficial

The results of protein folding studies with osmolytes can be highly variable depending on the experimental conditions in question. In one study, for example, researchers used guanidine hydrochloride to denature a common enzyme, lactate dehydrogenase, to its unfolded state, and, separately, they used strong acid to inactivate it. Unexpectedly, they found that both proline and the strong osmolyte TMAO inhibited, rather than promoted, the enzyme’s renaturation or reactivation.10

The authors noted, however, that the proline concentrations used were much higher than those estimated to accumulate in various organisms under physiological conditions. The high viscosity associated with these high concentrations probably inhibited the refolding. The authors also noted that a few other studies have shown a stabilizing effect of proline on the structure and function of lactate dehydrogenase.

Similar instances of role reversal—from protective osmolyte to denaturant—have been observed with proline and other osmolytes when used at very high concentrations.3 This underscores the need to be cognizant of the different ways in which different osmolytes can act on different proteins under different conditions. It’s also important to realize that combinations of osmolytes, as are typically found in nature, probably have additive and perhaps synergistic effects in their role as chemical chaperones. Supplementing with such combinations may have therapeutic value in human conformational disorders.

Bioengineering Is Natural—and Good

We end this discussion with an intriguing quote from an excellent paper on protein folding:6

We conclude this review with the recognition that the osmolyte effect is universal throughout all three kingdoms of life. A broad repertoire of biologically active osmolytes has been assembled via natural selection, enabling each organism to select for variants that are best suited to its cellular microenvironment and external conditions. Since Darwin, we have come to regard macroscopic characteristics, such as organelles or opposable thumbs, as the province of evolutionary biology. But osmolyte adaptation shows that natural selection is also at work on a strictly physicochemical level as well. Nature has been practicing successful bioengineering since the beginning of life on Earth.

And who is to say that we humans should not try to augment Nature’s efforts with some “bioengineering” of our own, e.g., by supplementing our diet with beneficial chemical compounds? In fact, who is to say that whatever we do on Earth is not, in a real sense, an aspect of Nature?

We are, after all, an integral part of the natural world, and, like every other organism, we are destined to affect it in various ways. At this stage of our evolution, as at every previous stage, it is simply natural for us to do what we do, whether the effects are considered to be good or bad. In terms of our ability to reshape our own health and longevity, in any case, the future looks bright.


  1. Fisher MT. Proline to the rescue. Proc Natl Acad Sci USA 2006;103(36):13265-6.
  2. Papp E, Csermely P. Chemical chaperones: mechanisms of action and potential use. Handb Exp Pharmacol 2006;172:405-16.
  3. Diamant S, Eliahu N, Rosenthal D, Goloubinoff P. Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem 2001;276(43):39586-91.
  4. Rösgen J, Pettitt BM, Bolen DW. Protein folding, stability, and solvation structure in osmolyte solutions. Biophys J 2005;89:2988-97.
  5. Street TO, Bolen DW, Rose GD. A molecular mechanism for osmolyte- induced protein stability. Proc Natl Acad Sci USA 2006;103(38):13997-4002.
  6. Bolen DW, Rose GD. Structure and energetics of the hydrogen-bonded backbone in protein folding. Annu Rev Biochem 2008;77:339-62.
  7. Pauling L. The Nature of the Chemical Bond and the Structure of Molecules and Crystals. Cornell University Press, Ithaca, NY, 1939.
  8. Kim SH, Yan YB, Zhou HM. Role of osmolytes as chemical chaperones during the refolding of aminoacylase. Biochem Cell Biol 2006;84:30-8.
  9. Ignatova Z, Gierasch LM. Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proc Natl Acad Sci USA 2006;103(36): 13357-61.
  10. Chilson OP, Chilson AE. Perturbation of folding and reassociation of lactate dehydrogenase by proline and trimethylamine oxide. Eur J Biochem 2003;270:4823-34.

Will Block is the publisher and editorial director of Life Enhancement magazine.

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