Protein Folding and the Hydrophobic Effect

MCAT Biochemistry Chapter 2 - Section 1.3 - Peptides and Proteins - Protein Folding and the Hydrophobic Effect

Sample MCAT Question - Hypothalamus and Pituitary Gland

Which hormone is not secreted by the anterior pituitary gland?

a) Growth Hormone

b) Endorphins

c) Vasopressin

d) Follicle-stimulating hormone

Click to Reveal the Answer

C is correct. Vasopressin.

The only hormone listed that is not secreted by the anterior pituitary gland is vasopressin, which is secreted by the posterior pituitary gland. Growth hormone, follicle-stimulating hormone, and endorphins, such as |alpha|-endorphin, |beta|-endorphin, and |gamma|-endorphin, are produced and secreted by the anterior pituitary gland.

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What is the Hydrophobic Effect?

In a previous post, we discussed how alpha helices and beta sheets fold together to form tertiary protein structures. As we mentioned, these tertiary structures are held together by all sorts of interactions, including hydrogen bonding between polar side chains, hydrophobic interactions between non-polar side chains, salt bridges that form between acidic and basic side chains, and disulfide bonds between thiol groups in cysteine side chains. However, apart from these side chain interactions, there is also another phenomenon that governs protein folding: the hydrophobic effect.

Broadly speaking, the hydrophobic effect is the tendency of non-polar molecules to aggregate together in an aqueous environment. The classic example is salad dressing: if you add oil to an aqueous substance like vinegar, you’ll notice that instead of dissolving, the oil forms large droplets or clumps, and if left alone, will eventually separate from the vinegar entirely.

The Hydrophobic Effect Explained

A common misconception is that the hydrophobic effect is the result of attractions between the non-polar molecules or some mysterious repulsive force between these molecules and water. In reality, however, the hydrophobic effect is the result of water’s attraction to itself. Let us explain.

Water molecules, as you probably know, favorably interact with each other via hydrogen bonding. This occurs because water is a polar molecule: the electron density surrounding the oxygen atom in a water molecule is greater than the electron density surrounding the two hydrogens. For this reason, the electron-poor, partially positive hydrogens in one water molecule will experience electrostatic attractions to electron-rich, partially negative oxygens in other water molecules. This electrostatic attraction is what’s known as a hydrogen bond.

Now, with this in mind, let’s consider what happens when we add some droplets of oil to water. The first thing to note is that the water doesn’t immediately dissolve the oil, as occurs with salts. This is because hydrogen bonding (and other types of electrostatic interactions) requires both participating molecules to be polar (or charged). Strong interactions do not therefore occur between the water and the non-polar molecules of oil. Instead, when we first combine the oil and water, the oil droplets will simply sit there, maintaining their globular shape, surrounded by water on all sides. This immediate layer of water surrounding the solute (in this case, the oil) is known as the solvation layer or solvation shell.

If we wait long enough, however, we’ll start see the oil droplets start to aggregate together, and eventually the oil and water will be completed separated in the container. This is the hydrophobic effect in action. But what accounts for it?

Well, we mentioned that the water molecules in the solvation layer cannot hydrogen bond with the oil they surround. This means that whenever oil is mixed into an aqueous environment, the hydrogen bonding in that aqueous environment is necessarily disrupted by an amount proportional to the surface area of the solvation layer. Think about it: when oil is mixed into water, some of those water molecules, which were previously engaged in favorable hydrogen bonding, are suddenly forced into the solvation layer interface with the oil, with which no hydrogen bonds are possible. Thus, whenever oil is introduced into water, the number of hydrogen bonds in that volume of water decreases by an amount proportional to the surface area of the solvation layer. This implies that when oil aggregates together, the disruption of water’s hydrogen bonding is minimized, since by clumping together, the surface area of the solvation layer is also minimized. This explains why oil aggregates in water; the oil aggregates because that way the surface area of the solvation layer is minimized, which in turn maximizes hydrogen bonding among the remaining water molecules. This lends meaning to our initial statement that the hydrophobic effect is the result of water’s attraction to itself.

We could, at this point, take the discussion one step further and ask why the oil-water system tends towards hydrogen-bond maximization. What’s so special about hydrogen bonding that makes the system spontaneously arrange to allow for more of it? The full answer, as is common, lies beyond the scope of the MCAT. For those interested, however, note that science isn’t actually quite sure why maximal hydrogen bonding is thermodynamically favorable: https://www.mdpi.com/1420-3049/27/20/7009/htm Some authorities think that minimizing the solvation layer increases entropy by releasing water molecules that would otherwise be trapped in static, non-hydrogen bonding positions surrounding the hydrophobic substance. Others think that hydrophobe aggregation is driven by a decrease in the system’s enthalpy (i.e. energy release) when more water molecules are allowed to engage in hydrogen bonding. Regardless, one thing is certain: the hydrophobic effect is driven by the thermodynamics of the surrounding water molecules, not the thermodynamics of the hydrophobic substance itself. Attractive forces between hydrophobic molecules are small and thus can’t explain the hydrophobic effect.

The Hydrophobic Effect & Protein Folding

Ok so, now that we have a basic grasp of the hydrophobic effect, let’s consider how it affects protein folding. Recall that proteins are molecular chains composed of individual amino acid residues. Also recall that some amino acids have non-polar hydrocarbon side chains. Just as oil separates from water, these non-polar side chains will tend to separate from the aqueous environment of the human body. Thus, when a particular protein begins to fold, the non-polar amino acid side chains will aggregate together in the interior of the protein, away from water. Other amino acids, which are either polar, acidic, or basic, will orient towards water. These tendencies, in addition to interactions between side chains, help give proteins their three-dimensional shape.

What’s interesting is that protein folding, in and of itself, isn’t thermodynamically favorable. A large macromolecule twisting and turning itself in a particular way would reduce the entropy, or disorder, of the system overall. However, if that macromolecule is surrounded by water and polar solutes in an aqueous environment, as is the case with proteins, the thermodynamic “pay off” of folding so that hydrophobic residues are hidden away from the aqueous medium is greater than the thermodynamic “cost” of the folding itself. Just as with the oil-water system we discussed early, the thermodynamic benefit of minimizing contact between hydrophobes and water is that it maximizes the favorable interactions between the water molecules themselves. When hydrophobic residues are clumped together away from water in the protein’s interior, the surrounding water molecules are allowed to go about their normal thermodynamic business, hydrogen bonding with each other and moving fluidly this way and that. If the hydrophobic residues were exposed, by contrast, all the fun would stop: a strict, rigid solvation layer would form around the protein and water-water hydrogen bonds would be disrupted. The protein folds in order to avoid this thermodynamically unfavorable possibility.

The crucial takeaway here is that the hydrophobic effect — whether we’re talking about oil and water or protein folding in the human body — is driven by the thermodynamics of the surrounding water, not the thermodynamics of hydrophobes themselves. If you pour olive oil onto the kitchen counter, it doesn’t magically clump together — the oil-oil interactions simply aren’t strong enough. But if you pour olive oil into water, shake the mixture up, and let it sit, just watch as the oil droplets slow clump together and form their own separate liquid phase. The water is the key player here, not the oil.