Proteins: Study Guide (Biology 1.7)

Proteins do most of the work in a cell

Enzymes that speed up reactions, channels that let ions cross membranes, antibodies that bind invaders, hormones that carry signals, and fibres that give skin and tendons their strength are all proteins. Each of these jobs depends on the protein having exactly the right shape. This topic explains where that shape comes from: a chain of building blocks that folds in a predictable way.

Amino acids: one shared core, one variable R group

The monomers of proteins are amino acids. Every amino acid has the same core. A central carbon atom is covalently bonded to four things:

1. a hydrogen atom;
2. an amine group, −NH₂;
3. a carboxyl group, −COOH;
4. a variable side chain called the R group.

Living things use about 20 common amino acids. They differ only in their R groups. You do not need to memorise any particular R group. You do need to sort R groups by their chemical properties, because those properties decide how each part of the chain behaves.

Type of R group	What it is like	Where it tends to end up in a protein dissolved in water	What it can do
Nonpolar (hydrophobic)	mostly C and H; no partial charges	buried inside, away from water	cluster with other nonpolar R groups (hydrophobic interactions)
Polar (hydrophilic)	partial charges (δ⁺ and δ⁻) but no overall charge	on the surface, in contact with water	form hydrogen bonds with water or with other polar R groups
Ionic (charged)	a full positive or negative charge	usually on the surface	attract an oppositely charged R group (ionic interaction); attract water

This is Topic 1.1 again. Water’s δ⁻ and δ⁺ ends attract polar and charged groups. Water molecules stay hydrogen-bonded to each other rather than to nonpolar groups, so nonpolar R groups are pushed together, away from water.

Peptide bonds: building the chain

Amino acids join by dehydration synthesis (Topic 1.3). The carboxyl group of one amino acid reacts with the amine group of the next. An −OH leaves the carboxyl group and an −H leaves the amine group. Together they form one water molecule. The carboxyl carbon of the first amino acid is now covalently bonded to the nitrogen of the second. This covalent bond is a peptide bond.

Notice three things in Figure 1.

• The R groups take no part in the peptide bond. They stick out from the chain, free to interact later.
• The new dipeptide still has a free amine group at one end and a free carboxyl group at the other. So the next amino acid can be added in the same way, giving a growing chain. The repeating N–C–C pattern along the chain is called the backbone.
• A single chain is a polypeptide. Every peptide bond releases one water molecule. For one chain of n amino acids there are n − 1 peptide bonds and n − 1 water molecules. Breaking a peptide bond is hydrolysis: water is added back, which is how digestion takes proteins apart.

Four levels of protein structure

A finished polypeptide does not stay as a floppy string. It folds into a precise shape. Biologists describe this shape at four levels.

Primary structure: the sequence

The primary structure is the order of amino acids in the chain, held together by peptide bonds. The sequence is set by the gene that codes for the protein (you will meet this in gene expression later in the course). The primary structure is the foundation for everything else: the specific sequence decides how the chain will fold and therefore the protein’s overall shape.

Secondary structure: local folding held by backbone hydrogen bonds

Stretches of the chain fold into regular local shapes. These are held by hydrogen bonds between atoms of the backbone: the δ⁻ oxygen of a C=O group on one amino acid is attracted to the δ⁺ hydrogen of an N–H group on another.

• In an α-helix, the chain coils like a spring. Each C=O forms a hydrogen bond with an N–H a few amino acids farther along the same stretch.
• In a β-pleated sheet, two or more stretches of the chain lie side by side. Hydrogen bonds form between neighbouring stretches, and the sheet has a folded, pleated look.

The key point: secondary structure comes from the backbone, not from the R groups. Most proteins contain some helices, some sheets and some less regular loops.

Tertiary structure: the whole chain folds, driven by R groups

The tertiary structure is the overall three-dimensional shape of one polypeptide. It results from interactions between R groups, which may be far apart in the sequence but end up close together after folding.

Interaction	Which R groups	What happens
Hydrogen bonds	polar R groups	δ⁺ and δ⁻ parts of two R groups attract
Hydrophobic interactions	nonpolar R groups	they cluster in the interior, away from water
Ionic interactions	ionic R groups with opposite charges	a positive R group attracts a negative one
Disulfide bridges	two R groups that contain sulfur	a covalent S–S bond forms between them

Hydrogen bonds, hydrophobic interactions and ionic interactions are individually weak, but there are many of them. A disulfide bridge is a covalent bond, so it is much stronger. It is the reason sulfur appears in your list of elements for proteins (Topic 1.2). For a protein dissolved in the cytoplasm, the usual result is a compact shape with nonpolar R groups buried in a core and polar or ionic R groups on the outside, facing water.

Quaternary structure: several chains together

Some proteins are made of two or more polypeptide chains. The way these chains (subunits) fit together is the quaternary structure. The chains are held together by the same kinds of interactions as in tertiary structure. Hemoglobin, which carries oxygen in red blood cells, has four polypeptide chains: two of one kind (α) and two of another (β). Collagen, a structural protein in skin and tendons, is made of three chains wound around each other. A protein made of only one chain has no quaternary structure.

Shape determines function

All four levels together decide what a protein can do. An enzyme works because its active site has the right shape and the right R groups to bind its substrate. A channel protein works because its chains surround a pore of the right size. So you can follow a chain of cause and effect:

sequence → folding → shape → function

This chain lets you predict the effect of a change, which is the main skill this topic tests.

• A change in sequence. In sickle cell disease, one amino acid in each β chain of hemoglobin is different: glutamic acid, which has a charged (ionic) R group, is replaced by valine, which has a nonpolar R group. The new nonpolar patch is on the outside of the protein. When oxygen levels are low, hemoglobin molecules stick to each other through these patches and form long fibres. The red blood cells become stiff and sickle-shaped and can block small blood vessels. One amino acid out of more than a hundred is enough to change the function.
• Not every change matters equally. Replacing one polar R group on the surface with another polar R group often has little effect. Replacing a buried nonpolar R group with an ionic one usually disrupts the fold, because the charged group is now trapped away from water.
• A change in conditions. High temperature or a change in pH can disrupt the weak interactions that hold secondary, tertiary and quaternary structure. The protein loses its shape and usually its function. This is denaturation, which you will study with enzymes in Unit 3. Denaturation usually leaves the peptide bonds, and so the primary structure, intact.

Worked example 1: counting bonds and finding the mass of a protein

Question. A fictional enzyme is made of two polypeptide chains: chain A has 96 amino acids and chain B has 128. Take the mean mass of a free amino acid as 128 Da and the mass of water as 18 Da. (A dalton, Da, is the unit for the mass of molecules; one hydrogen atom has a mass of about 1 Da.)

(a) How many peptide bonds are in the enzyme? (b) How many water molecules were released in making it? (c) Estimate the mass of the enzyme.

1. Peptide bonds per chain. A chain of n amino acids has n − 1 peptide bonds. Chain A: 96 − 1 = 95. Chain B: 128 − 1 = 127.
2. Total peptide bonds. 95 + 127 = 222. The chains are separate polypeptides, so you subtract 1 for each chain, not once for the whole protein.
3. Water released. One water molecule per peptide bond: 222 water molecules.
4. Mass of the free amino acids. Total amino acids = 96 + 128 = 224. Mass = 224 × 128 Da = 28 672 Da.
5. Mass of water lost. 222 × 18 Da = 3996 Da.
6. Mass of the enzyme. 28 672 − 3996 = 24 676 Da, about 24.7 kDa (1 kDa = 1000 Da).

Check. Each amino acid in the protein now has a mean mass of 24 676 ÷ 224 ≈ 110 Da. That is less than 128 Da, as it should be, because most amino acids have lost the mass of one water molecule.

Common errors. Treating both chains as one gives 223 bonds and 24 658 Da. Using one water per amino acid gives 224 and 24 640 Da. Both errors look small here, but they show a misunderstanding of how peptide bonds form. If the chains are also linked by a disulfide bridge, two hydrogen atoms are lost, about 2 Da, which you can ignore at this precision.

Worked example 2: predicting the effect of changing one amino acid

Question. In a fictional bacterial enzyme, a positively charged R group at position 58 forms an ionic interaction with a negatively charged R group at position 112. A mutation replaces the amino acid at position 58 with one that has a negatively charged R group. Researchers measured the activity of each enzyme at 25 °C, then heated samples to 50 °C for 10 minutes and measured the percentage of activity that remained.

Enzyme	Activity at 25 °C (units)	Activity remaining after heating: trial 1 / 2 / 3 (%)
Normal (wild type)	52.0	86 / 90 / 88
Mutant (position 58 changed)	50.4	34 / 29 / 30

(a) Predict the effect of the mutation on the enzyme’s structure. Justify your prediction. (b) Calculate the mean activity remaining for each enzyme. (c) Use the data to evaluate your prediction.

(a) Prediction.

1. Identify the change: a positive R group becomes negative. The primary structure has changed at one position.
2. Identify the interaction lost: R groups 58 and 112 are now both negative. Like charges repel, so the ionic interaction is lost.
3. Link to the level of structure: this ionic interaction helps hold two parts of the chain together, so it is part of the tertiary structure.
4. Predict: the folded shape will be less stable. It may still fold, because many other interactions remain, but it should unfold more easily when extra energy (heat) is added.

(b) Calculation.

• Normal: (86 + 90 + 88) ÷ 3 = 264 ÷ 3 = 88.0%.
• Mutant: (34 + 29 + 30) ÷ 3 = 93 ÷ 3 = 31.0%.
• The mutant kept 31.0 ÷ 88.0 × 100 ≈ 35% as much activity as the normal enzyme did after heating.

(c) Evaluation. At 25 °C the two enzymes have almost the same activity (50.4 ÷ 52.0 ≈ 97%). So the mutant still folds into a working shape at normal temperature, and the active site is probably not where the change is. After heating, the mutant lost far more activity, and the ranges do not overlap (86–90% against 29–34%). This supports the prediction that the lost ionic interaction makes the tertiary structure less stable. It does not prove it: only three trials were done, and the experiment does not show the structure directly. A stronger test would compare a third enzyme in which both charges are reversed (position 58 negative and position 112 positive), which should restore the attraction and the heat stability.

Common misconceptions

• “Amino acids join through their R groups.” No. The peptide bond forms between the carboxyl group of one amino acid and the amine group of the next. The R groups stick out and are free to interact.
• “A peptide bond is a hydrogen bond.” A peptide bond is a strong covalent bond in the backbone. Hydrogen bonds are the weak attractions that shape secondary and tertiary structure.
• “Secondary structure comes from R-group interactions.” Helices and sheets are held by hydrogen bonds between backbone atoms. R-group interactions build the tertiary structure.
• “Hydrophobic interactions are strong bonds between nonpolar groups.” Nonpolar R groups cluster because water molecules hydrogen-bond with each other and with polar groups, which leaves the nonpolar groups together in the interior.
• “Every protein has four levels of structure.” Only proteins with two or more chains have quaternary structure.
• “One amino acid out of hundreds cannot matter.” It can, if it changes an important interaction, as sickle cell hemoglobin shows. It can also have almost no effect if the new R group has similar properties.
• “Denaturation breaks the protein into amino acids.” Denaturation disrupts the folded shape. Breaking peptide bonds is hydrolysis, a different process.
• “A polypeptide of n amino acids has n peptide bonds.” It has n − 1 per chain, and the same number of water molecules is released.

Where this leads

This is the last topic of Unit 1. In Topic 1.6, Nucleic Acids, you saw that DNA stores information as a sequence. Later in the course you will see how that sequence is used to build the amino acid sequence of a protein. Proteins then appear everywhere: membrane proteins in Unit 2, enzymes and denaturation in Unit 3, and signalling proteins in Unit 4. Use the course roadmap to plan your next unit. Test yourself now with the practice questions, then use the revision notes and the checklist to consolidate.