Protein folding and function

From a chain to a machine

The free course on proteins showed that a protein is a chain of amino acids joined by peptide bonds. A chain is not yet a machine. An enzyme works only when its chain is folded into one exact shape.

This lesson asks four questions. Why does a chain fold? What happens when it unfolds? How can one change in the chain ruin the shape? And how does the charge of a protein depend on pH?

Why a chain folds

Weak forces do the folding. Each one is feeble on its own, but a chain has hundreds of them, and together they hold the shape.

• Hydrophobic collapse. The side chains of leucine, valine and phenylalanine avoid water. In a cell they bury themselves together in the middle of the protein, and the polar side chains stay on the outside. This is the strongest push towards folding.
• Hydrogen bonds. The N–H and C=O groups of the backbone bond to each other. They make the alpha helix and the beta sheet.
• Ionic bonds. A positive side chain, such as lysine, attracts a negative one, such as aspartate. The pair is a salt bridge.
• Disulphide bridges. Two cysteine side chains join in an S–S bond. This is a true covalent bond, much stronger than the others. It is common in proteins that leave the cell, such as insulin.
• Van der Waals attractions pack the core tightly.

The order of the amino acids, the primary structure, holds all the information. Christian Anfinsen showed this with the enzyme ribonuclease. Urea and a thiol reagent unfolded it, and it lost its activity. When he removed both chemicals slowly, the chain refolded and the enzyme worked again.

Folding is guided

A chain could try a huge number of shapes. If each of 100 links could sit in just three positions, the chain would have $3^{100}$ shapes, about $5 \times 10^{47}$. A chain that tried them all in turn would never finish. Real proteins fold in a fraction of a second to a few seconds, because folding runs downhill through a few steps.

A cell is crowded, and a half-folded chain has sticky hydrophobic patches. Chaperones protect new chains. Heat shock proteins such as Hsp70, and the barrel-shaped GroEL, stop chains from clumping and give them a chamber in which to fold. Cells make more of them when the temperature rises, which is how they got their name.

One change in the chain, one change in the shape

In the beta chain of haemoglobin, the sixth amino acid is glutamic acid. In sickle-cell haemoglobin it is valine. Glutamic acid is polar and charged. Valine is hydrophobic.

The new sticky patch lies on the surface. When oxygen is low, the molecules stick together in long fibres, and the red cell bends into a sickle. One amino acid in about 146 changes the shape, and the shape decides the function.

Denaturation

Denaturation is the loss of the folded shape. The peptide bonds stay whole. Only the weak bonds, and sometimes the disulphide bridges, give way.

• Heat shakes the weak bonds apart. In a boiled egg the unfolded albumin chains stick to each other through their exposed hydrophobic patches. The white turns solid, and cooling does not undo it.
• pH. Strong acid or alkali changes the charges on the side chains, so salt bridges and hydrogen bonds fail.
• Urea competes for hydrogen bonds and helps water dissolve hydrophobic groups, so the core opens.
• A thiol such as mercaptoethanol breaks disulphide bridges.

Some denaturation is reversible, as with ribonuclease. Some is not, as with the egg. An enzyme that has lost its shape has lost its active site, and it cannot work.

Charge, pH and the isoelectric point

Amino acid groups gain or lose a proton as the pH changes. A carboxyl group is neutral in acid and negative in alkali. An amino group is positive in acid and neutral in alkali. So the net charge of an amino acid, or of a protein, changes with pH.

The pH at which the net charge is zero is the isoelectric point, written pI. For a small amino acid, pI is the average of the two pK values on either side of the neutral form.

• Glycine has pK values 2.3 (carboxyl) and 9.6 (amino). Its pI is $\dfrac{2.3 + 9.6}{2} = 5.95$, about 6.0.
• Aspartic acid has an acidic side chain. Its pK values are 1.9, 3.7 and 9.6. The neutral form lies between 1.9 and 3.7, so pI is 2.8.
• Lysine has a basic side chain. Its pK values are 2.2, 9.0 and 10.5. The neutral form lies between 9.0 and 10.5, so pI is about 9.8.

Move the acid pK up and down. The curve slides, and the dot moves with the average of the two pK values.

Below its pI a protein carries a net positive charge. Above its pI the charge is negative. This has three uses.

• Solubility. At the pI the charges cancel, the molecules do not repel each other, and the protein is least soluble. Casein, the main protein of milk, has a pI near 4.6. Milk curdles when bacteria make lactic acid and the pH falls to about 4.6.
• Electrophoresis. In an electric field a positive protein moves towards the negative electrode, and a negative protein moves towards the positive one. A protein at its pI does not move.
• Separation. Chemists choose a pH between the pI values of two proteins, so that one is positive and the other is negative.

Try it in Python

The program finds the net charge of three amino acids at three pH values. It then scans the pH to find where each charge is zero.

Practise with new numbers

Common mistakes

• Saying that denaturation breaks peptide bonds. It does not. The chain stays whole, and only the folded shape is lost.
• Calling a disulphide bridge a weak bond. It is covalent. It is the strongest link inside a folded protein.
• Averaging the wrong pK values. For aspartic acid the neutral form lies between 1.9 and 3.7, not between 1.9 and 9.6.
• Saying that a protein at its pI has no charged groups. The net charge is zero. Many groups still carry a charge.
• Saying that hydrophobic groups attract each other. Water pushes them together, which is the hydrophobic effect.

Remember these

• Folding is driven by the hydrophobic effect, and held by hydrogen bonds, salt bridges, van der Waals forces and disulphide bridges.
• The primary structure holds the information for the fold. Chaperones help the chain fold without clumping.
• Heat, pH, urea and thiols denature a protein. Peptide bonds survive.
• pI is the pH of zero net charge. A protein is least soluble there, and it does not move in an electric field.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Name two kinds of bond that heat breaks in a protein, and one that it does not.

2. Why does a chaperone help a new chain in a crowded cell?

3. A protein has a pI of 7.4. Is it positive or negative in a buffer of pH 6.0, and which electrode does it move towards?

4. Why is a disulphide bridge more common in a protein that leaves the cell than in one that stays inside?

5. In sickle-cell haemoglobin a charged residue is replaced by a hydrophobic one. Why does that make the molecules stick together?