Lesson 1.7 · Unit 1 · CED topic 1.7
Proteins
If DNA is the instruction manual, proteins are the workers. Enzymes speed up reactions, hemoglobin carries oxygen, collagen holds skin together, antibodies recognize invaders, and channels in membranes let specific substances through. A cell contains thousands of different proteins, and they are all built from the same 20 kinds of amino acid. What makes one protein an enzyme and another a structural fiber is the order of its amino acids and, as a result, the three-dimensional shape it folds into.
Describe the structure and function of proteins. The CED states that the molecular structure of the amino acids is beyond the scope of the exam. You should know the parts they share, the three kinds of R group, and the four levels of structure.
Amino acids and the peptide bond
Every amino acid has a central carbon bonded to four things: a hydrogen atom, a carboxyl group (−COOH), an amino group (−NH2), and a variable R group. The R group is the only difference among the 20 amino acids. Amino acids are linked by peptide bonds, covalent bonds between the carboxyl group of one amino acid and the amino group of the next. This is dehydration synthesis (lesson 1.3), so each peptide bond releases one water. A chain of amino acids is a polypeptide, with an N-terminus (free amino group) at one end and a C-terminus (free carboxyl group) at the other.
The R groups fall into three categories by their chemical properties, and these properties decide how a protein folds.
| R-group category | Property | Examples | Where it tends to end up in a folded protein |
|---|---|---|---|
| Hydrophobic (nonpolar) | No charges; avoids water | Valine, leucine, alanine | Inside the protein, away from water |
| Hydrophilic (polar) | Partial charges; hydrogen-bonds with water | Serine, threonine | On the surface, in contact with water; or hydrogen-bonded inside |
| Ionic (charged) | Full positive or negative charge | Glutamate (−), lysine (+) | On the surface; opposite charges can form ionic bonds with each other |
The R groups on the amino acids determine the structure and function of that region of the protein. The sequence of amino acids is called the protein's primary structure (EK 1.7.A.3), and it determines the overall shape.
Four levels of structure
- Primary: the linear sequence of amino acids, held together by covalent peptide bonds.
- Secondary: local folding of the chain into shapes such as the α helix and the β pleated sheet. These are held by hydrogen bonds between atoms of the polypeptide backbone (not the R groups).
- Tertiary: the overall three-dimensional shape of one polypeptide. It forms through interactions between R groups: hydrogen bonds, hydrophobic interactions, ionic interactions, and disulfide bridges (covalent S–S bonds between the sulfhydryl groups of two cysteines).
- Quaternary: the arrangement of two or more polypeptides into one functional protein. Hemoglobin, with four chains, is an example. A protein made of a single chain has no quaternary structure.
All four levels together determine the function of the protein. A change at the lowest level can ripple upward: alter one amino acid in the sequence and the folding, the shape, and the function can all change. That is the cause-and-effect reasoning the exam asks you to practice (“predict the causes or effects of a change in a biological system”).
Denaturation: losing the shape
The weak interactions that hold secondary, tertiary, and quaternary structure are easily disrupted. Heat, a change in pH, or certain chemicals can break them, and the protein unfolds. This is denaturation. The primary structure is not affected, because the peptide bonds are covalent and strong, but without its shape the protein cannot function. Cooking an egg is denaturation: the clear proteins unfold and tangle into a white solid, and the change is not reversed when the egg cools. Breaking the peptide bonds themselves is a different process, hydrolysis (lesson 1.3).
Samples of an enzyme were kept for 10 minutes at different temperatures, then cooled to 25 °C and tested for activity. Activity is given as a percentage of the 25 °C sample. Describe the pattern, explain it, and predict the result for the same enzyme from a hot-spring bacterium.
| Temperature of 10 min exposure (°C) | 25 | 40 | 50 | 60 | 70 | 80 |
|---|---|---|---|---|---|---|
| Activity measured at 25 °C (% of control) | 100 | 98 | 85 | 30 | 4 | 0 |
Describe. Activity stays near 100% up to 40 °C, then falls: 85% at 50 °C, 30% at 60 °C, 4% at 70 °C, and none at 80 °C. The steepest drop is between 50 and 60 °C (55 percentage points).
Explain. Higher temperature makes the atoms move faster until the weak hydrogen bonds, ionic bonds, and hydrophobic interactions that hold the enzyme's folded shape break. The protein unfolds (denatures) and its active site no longer has the correct shape, so it cannot bind its substrate. The peptide bonds are covalent, so the primary structure stays intact.
Because the activity was measured after cooling to 25 °C, the low values show that the enzyme did not refold, so the damage was not reversible in this case.
Predict. An enzyme from a hot-spring bacterium would keep its activity at higher temperatures, with the steep drop shifted well above 60 °C. Its amino acid sequence differs, so it has extra stabilizing interactions (for example more ionic interactions or disulfide bridges) that hold the shape together at high temperature.
In the 1960s Christian Anfinsen studied ribonuclease, an enzyme with 124 amino acids and four disulfide bridges. He treated it with urea (which disrupts hydrogen bonds and hydrophobic interactions) and β-mercaptoethanol (which breaks disulfide bridges). The enzyme unfolded and lost nearly all activity. When he removed both chemicals, the enzyme refolded and regained nearly all of its original activity.
Question. What does the experiment show about how a protein gets its shape? Claim: the amino acid sequence contains the information that determines the final folded shape. Evidence: the enzyme refolded correctly after the denaturing chemicals were removed, with no other cell machinery present, and the only thing that was never changed was the amino acid sequence (the peptide bonds were never broken). Reasoning: if the sequence were not enough, a chain that was unfolded in a test tube could not find its correct shape on its own. A supporting detail: with eight cysteines there are \(7 \times 5 \times 3 \times 1 = 105\) ways to pair them into four disulfide bridges, and only one gives an active enzyme. A random pairing would be correct less than 1 percent of the time, yet the refolded enzyme was almost fully active.
Hemoglobin carries oxygen in red blood cells. It has four polypeptides, two α chains and two β chains. In sickle-cell disease, the sixth amino acid in the β chain is valine instead of glutamate. Predict the effect on the protein and justify it.
Identify the change. Glutamate has an acidic, negatively charged R group, so it is hydrophilic and sits happily on the surface of the protein in contact with water. Valine has a nonpolar, hydrophobic R group.
Predict. The substitution creates a hydrophobic spot on the outside of the protein, where there used to be a charged one.
Justify. Hydrophobic regions avoid water and attract other hydrophobic regions. In low-oxygen conditions the exposed valine of one hemoglobin molecule fits into a hydrophobic pocket on another, so the molecules stick together and form long rigid fibers (a change in how the subunits interact). The fibers distort red blood cells into a sickle shape, which can block small blood vessels.
One amino acid out of about 146 in the chain changes the behavior of the whole protein, because the primary structure sets everything above it.
There are 20 possible amino acids at each position, so the number of different sequences of length \(n\) is \(20^{n}\). For \(n = 10\) that is \(20^{10} = 1.024 \times 10^{13}\) different peptides; for a 100-amino-acid protein it is \(20^{100} \approx 1.3 \times 10^{130}\). A linear chain of \(n\) amino acids has \(n-1\) peptide bonds. The diversity of protein sequences is what lets a few kinds of subunit make an unlimited variety of molecules.
- Name the bond for each level. Primary: peptide bonds. Secondary: hydrogen bonds between backbone atoms. Tertiary: R-group interactions (hydrogen, hydrophobic, ionic, disulfide). Quaternary: interactions between polypeptides.
- Denaturation affects secondary, tertiary, and quaternary structure, not the primary. It does not break peptide bonds.
- When you predict the effect of a mutation or change in pH, trace the chain: R group changed, interactions changed, shape changed, function changed.
- Say whether an amino acid R group is hydrophobic, polar, or ionic, and where it would sit in the folded protein.
- Not every protein has quaternary structure.
- Match each type of bond or interaction with the level of protein structure it stabilizes: (a) peptide bond; (b) hydrogen bonds between the backbone N–H and C=O groups; (c) disulfide bridge between two cysteines; (d) interactions between two separate polypeptide chains.
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(a) Primary structure. (b) Secondary structure (α helix and β pleated sheet). (c) Tertiary structure. (d) Quaternary structure.
- Insulin is made of two polypeptides, one with 21 amino acids and one with 30. How many peptide bonds does it contain?
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The 21-residue chain has \(21 - 1 = 20\) peptide bonds and the 30-residue chain has \(30 - 1 = 29\), so \(20 + 29 = 49\) in total.
- How many different peptides of length 5 could be made from the 20 amino acids? Why is this relevant to the variety of proteins?
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\(20^{5} = 3{,}200{,}000\). Even a very short chain can be arranged in millions of ways; real proteins are hundreds of amino acids long, so the number of possible sequences is far larger than the number of proteins that exist. The order of the amino acids gives each protein its own shape and function.
- A mutation replaces a leucine buried in the hydrophobic core of an enzyme with aspartate, an amino acid with a negatively charged R group. Predict the effect on the enzyme and justify it.
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Prediction: the enzyme would misfold or become unstable and lose most of its activity. Justification: leucine is nonpolar and was held in the core by hydrophobic interactions away from water. Aspartate is charged and hydrophilic and does not fit in a nonpolar interior, so the hydrophobic interactions are disrupted. The tertiary structure changes, the active site changes shape, and the enzyme can no longer bind its substrate.
- Adding lemon juice (acid) to milk causes the milk to curdle. Explain this in terms of protein structure.
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The acid lowers the pH, which adds hydrogen ions to the R groups of the milk proteins and changes their charges. That disrupts the ionic interactions and hydrogen bonds that hold the proteins in their folded shapes, so they denature, unfold, and clump together as curds. The peptide bonds are not broken, so the primary structure stays the same.
- A student states: “Heating a protein breaks its peptide bonds, which is why it stops working.” Evaluate this statement.
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The statement is incorrect. Ordinary heating denatures a protein by breaking the weak hydrogen bonds, ionic interactions, and hydrophobic interactions that hold its secondary, tertiary, and quaternary structure. The covalent peptide bonds, which make up the primary structure, stay intact. The protein stops working because it has lost its shape, not because the chain has been cut.