Proteins

AS · 12 min

Proteins do almost everything in a cell: they catalyse reactions (enzymes), carry oxygen (haemoglobin), defend the body (antibodies), carry signals (receptors and some hormones), move substances across membranes (channels and carriers) and hold tissues together (collagen). Each protein can do its job only because it has a precise three-dimensional shape, and that shape is determined by its sequence of amino acids. This note builds a protein from the amino acid up through the four levels of structure, and explains the bonds that hold it in shape. It underpins enzymes, membranes, haemoglobin, antibodies and mutations, so it is one of the most important notes in the course.

Amino acids

Proteins are polymers of amino acids. Twenty different amino acids are used in the proteins of living organisms, and they all share the same basic structure.

Key result

General structure of an amino acid

A central carbon atom bonded to four groups:

an amine group, $-\text{NH}_2$ ;
a carboxyl group, $-\text{COOH}$ ;
a hydrogen atom, $-\text{H}$ ;
an R group (side chain), which is different in each of the 20 amino acids.

\text{H}_2\text{N} - \underset{\displaystyle\text{R}}{\overset{\displaystyle\text{H}}{\underset{|}{\overset{|}{\text{C}}}}} - \text{COOH}

The R group gives each amino acid its properties. The simplest is glycine, where R is just a hydrogen atom. R groups can be:

Type of R group	Examples	Behaviour
non-polar, hydrophobic	alanine ( $-\text{CH}_3$ ), valine, leucine, phenylalanine	repelled by water; cluster together inside proteins
polar, uncharged	serine ( $-\text{CH}_2\text{OH}$ ), threonine, asparagine	form hydrogen bonds; hydrophilic
positively charged (basic)	lysine ( $-\text{NH}_3^+$ at the end), arginine	form ionic bonds with negative R groups; hydrophilic
negatively charged (acidic)	aspartic acid, glutamic acid ( $-\text{COO}^-$ )	form ionic bonds with positive R groups; hydrophilic
containing sulfur	cysteine ( $-\text{CH}_2\text{SH}$ )	two cysteines can form a disulfide bond

You do not need to learn the R groups of specific amino acids, but you must be able to work out from a given R group what kinds of interaction it could form.

The peptide bond

Two amino acids join in a condensation reaction between the carboxyl group of one and the amine group of the other. An –OH is removed from the carboxyl group and an –H from the amine group, forming a molecule of water, and a covalent peptide bond ( $-\text{CO–NH}-$ ) links the two amino acids.

\text{H}_2\text{N–CHR}_1\text{–COOH} + \text{H}_2\text{N–CHR}_2\text{–COOH} \longrightarrow \text{H}_2\text{N–CHR}_1\text{–CO–NH–CHR}_2\text{–COOH} + \text{H}_2\text{O}

Two amino acids form a dipeptide.
Many amino acids form a polypeptide. A polypeptide always has a free amine group at one end (the N-terminus) and a free carboxyl group at the other (the C-terminus).
A protein consists of one or more polypeptides folded into a specific 3-D shape.

Hydrolysis breaks peptide bonds by adding water. In digestion this is catalysed by protease enzymes; in the laboratory, by boiling with concentrated acid.

Drawing the formation of a peptide bond

Draw two amino acids side by side, with the carboxyl group of the left one next to the amine group of the right one.
Ring the –OH of the carboxyl group and one –H of the amine group.
Show them leaving as $\text{H}_2\text{O}$ .
Draw the product with C (double-bonded to O) joined directly to N (bonded to H). Label the C–N bond "peptide bond".
Label the reaction "condensation".

Counting bonds and water (routine)

A polypeptide contains 146 amino acids. (a) How many peptide bonds does it contain? (b) How many water molecules are needed to hydrolyse it completely into amino acids?

Solution

(a) In a chain of $n$ amino acids there are $n - 1$ peptide bonds: $146 - 1 = 145$ .

(b) One water molecule is used for each bond hydrolysed: 145 water molecules.

(If a protein has more than one polypeptide chain, count $n - 1$ for each chain. Haemoglobin, with four chains of 141, 141, 146 and 146 amino acids, has $140 + 140 + 145 + 145 = 570$ peptide bonds.)

Four levels of protein structure

Definition

Primary structure: the sequence of amino acids in a polypeptide chain, held together by peptide bonds.
Secondary structure: the regular, repeated folding or coiling of the polypeptide chain into an α-helix or a β-pleated sheet, held in place by hydrogen bonds between the $-\text{C=O}$ group of one amino acid and the $-\text{N–H}$ group of another amino acid further along the chain.
Tertiary structure: the further folding of the whole polypeptide (including its secondary structures) into a precise, compact three-dimensional shape, held in place by interactions between R groups: hydrophobic interactions, hydrogen bonds, ionic bonds and disulfide bonds.
Quaternary structure: the association of two or more polypeptide chains to form a functional protein (sometimes with non-protein prosthetic groups), held together by the same types of interaction as the tertiary structure.

Primary structure

The primary structure is the order of amino acids, determined by the sequence of bases in the gene that codes for the polypeptide. With 20 amino acids, the number of possible sequences is enormous: for a chain of just 100 amino acids there are $20^{100}$ possibilities. A change of a single amino acid can change the way the chain folds and so change its tertiary structure and function. (This is the basis of sickle cell anaemia; see Gene mutations.)

Secondary structure

The backbone of a polypeptide ( $-\text{N–C–C–N–C–C}-$ ) contains slightly negative oxygen atoms in the $\text{C=O}$ groups and slightly positive hydrogen atoms in the $\text{N–H}$ groups. These form hydrogen bonds with each other, folding the chain in one of two regular ways:

α-helix: the chain coils into a spiral, with each $\text{C=O}$ hydrogen-bonded to the $\text{N–H}$ of the amino acid four places along. The R groups point outwards.
β-pleated sheet: lengths of chain lie side by side (running in the same or opposite directions), joined by hydrogen bonds between them, forming a pleated, sheet-like structure.

Secondary structure involves only the backbone, not the R groups. Individually, hydrogen bonds are weak, but there are so many that the structures are stable. Parts of a polypeptide may have no regular secondary structure at all.

Tertiary structure

The polypeptide then folds further into a specific, compact 3-D shape, which depends on the interactions between the R groups. Because those interactions depend on which R groups are present and where, the tertiary structure is determined by the primary structure.

Quaternary structure

Many proteins consist of more than one polypeptide. Haemoglobin has four polypeptides (two α-globin and two β-globin chains), each with a haem group; collagen has three polypeptides twisted together; antibodies have four (two heavy and two light chains). Proteins with only one polypeptide, such as myoglobin and lysozyme, have no quaternary structure.

Interactions that hold proteins in shape

Key result

Interaction	Between	Strength	Broken by
Hydrophobic interactions	non-polar (hydrophobic) R groups, which cluster together in the interior of the protein, away from water	individually weak	high temperature; organic solvents
Hydrogen bonds	slightly positive H (in –OH or –NH groups) and slightly negative O or N; between R groups (tertiary) or backbone C=O and N–H (secondary)	weak individually, but very numerous	high temperature; pH changes
Ionic bonds	oppositely charged R groups, e.g. $-\text{NH}_3^+$ and $-\text{COO}^-$	stronger than hydrogen bonds, but weakened by water	changes in pH (which alter the charges on R groups); high temperature
Covalent bonds, including disulfide bonds	sulfur atoms of two cysteine R groups, forming $-\text{S–S}-$ (disulfide bridges); peptide bonds are also covalent	strong	not broken by moderate heat; broken by reducing agents

Hydrophobic interactions deserve special attention. When a polypeptide folds in water, its hydrophobic R groups are pushed together into the centre of the molecule, where they are shielded from water, and hydrophilic R groups end up on the outside, in contact with water. This is the main reason globular proteins are soluble in water: their surface is covered in hydrophilic R groups.

Denaturation

Denaturation is a change in the three-dimensional (tertiary and quaternary) shape of a protein, caused by breaking the hydrogen bonds, ionic bonds and hydrophobic interactions that hold it in shape. The primary structure is not changed: peptide bonds are not broken. Because function depends on shape, a denatured protein loses its function; for example, a denatured enzyme no longer has an active site complementary to its substrate.

High temperature increases the kinetic energy of the molecule so that it vibrates more, breaking hydrogen bonds and other weak interactions.
Changes in pH alter the charges on R groups (by adding or removing $\text{H}^+$ ), disrupting ionic bonds and hydrogen bonds.

Denaturation is usually irreversible. Disulfide bonds are not broken by heat, which helps proteins with many disulfide bonds (like those in hair keratin) keep their shape.

Globular and fibrous proteins

Key result

Globular proteins are generally soluble and have physiological (metabolic) roles. Fibrous proteins are generally insoluble and have structural roles.

Feature	Globular proteins	Fibrous proteins
Shape	compact, roughly spherical, folded tertiary structure	long, thin strands or sheets
Solubility in water	generally soluble (hydrophilic R groups on the outside)	generally insoluble (many hydrophobic R groups on the surface)
Role	physiological / metabolic: enzymes, antibodies, transport proteins, hormones, receptors	structural: support, strength, protection
Primary structure	varied sequence of many different amino acids	often a repetitive sequence
Sensitivity to heat and pH	easily denatured	more stable
Examples	haemoglobin, enzymes (amylase, catalase), antibodies, insulin	collagen, keratin, elastin

The two syllabus examples, haemoglobin (globular) and collagen (fibrous), are covered in Haemoglobin and collagen.

Predicting interactions from R groups

A polypeptide contains, among others, the following amino acids: cysteine (R $= -\text{CH}_2\text{SH}$ ) at positions 12 and 58; lysine (R ends in $-\text{NH}_3^+$ ) at position 30; glutamic acid (R ends in $-\text{COO}^-$ ) at position 77; valine and leucine (non-polar R groups) at positions 40 and 65; serine (R $= -\text{CH}_2\text{OH}$ ) at position 90.

(a) Name the interaction that could form between: (i) positions 12 and 58; (ii) positions 30 and 77; (iii) positions 40 and 65. (b) Suggest where in the folded protein valine and leucine are likely to be found. (c) Predict the effect of lowering the pH on the interaction in (a)(ii).

Solution

(a) (i) A disulfide bond (covalent) between the sulfur atoms of the two cysteines. (ii) An ionic bond between the positively charged lysine R group and the negatively charged glutamic acid R group. (iii) Hydrophobic interactions between the non-polar R groups.

(b) In the interior of the protein, away from water, since they are hydrophobic.

(c) At low pH there are many $\text{H}^+$ ions; the $-\text{COO}^-$ of glutamic acid gains $\text{H}^+$ to become $-\text{COOH}$ , which is uncharged, so the ionic bond breaks. The tertiary structure changes (the protein may denature).

Explain the levels of structure (6 marks)

Explain what is meant by the primary, secondary and tertiary structure of a protein, naming the bonds involved at each level.

Solution

Primary: the sequence (order) of amino acids in the polypeptide;
held by peptide bonds (covalent).
Secondary: regular folding/coiling into α-helix or β-pleated sheet;
held by hydrogen bonds between C=O and N–H groups of the backbone (amino acids at different points in the chain).
Tertiary: further folding of the polypeptide into a specific/precise 3-D shape;
held by interactions between R groups: hydrogen bonds, ionic bonds, disulfide bonds and hydrophobic interactions.

Note that for secondary structure, the examiners want hydrogen bonding within the backbone (between –CO and –NH groups), not between R groups.

Exam-hard: linking sequence to function

An enzyme has 320 amino acids. A mutation changes one amino acid in the chain from a positively charged amino acid to a non-polar one. Suggest how this single change could cause the enzyme to stop working, and why some other single changes have no effect.

Solution

The change alters the primary structure (one R group is different).
The original positively charged R group may have formed an ionic bond with a negatively charged R group; this bond can no longer form.
A non-polar R group may instead form hydrophobic interactions, so the polypeptide folds differently.
This changes the tertiary structure and therefore the shape of the active site.
The substrate no longer fits / is no longer complementary, so no enzyme–substrate complexes form.
Other changes may have no effect if the new amino acid has similar properties (similar R group), or if the amino acid is on the surface far from the active site and not involved in bonds that hold the shape.

Watch out

The primary structure is the sequence of amino acids, not "the amino acids" or "the chain". Use the word sequence (or order).
In secondary structure, hydrogen bonds form between the C=O and N–H groups of the backbone, not between R groups.
Denaturation does not break peptide bonds. Writing "heat breaks the peptide bonds" loses the mark.
Disulfide bonds are between cysteine R groups (sulfur atoms) and are covalent: do not call them "sulfur bonds" or "disulfide bridges between any amino acids".

Exam tip

Learn the four interactions by their syllabus names: hydrophobic interactions, hydrogen bonding, ionic bonding, covalent bonding including disulfide bonds.
In explanation questions, connect the chain: primary structure → R groups and their positions → bonds formed → tertiary shape → function (active site, binding site, solubility).
"State" the property of globular proteins in syllabus wording: soluble, physiological roles; fibrous: insoluble, structural roles.
The biuret test detects proteins because it reacts with peptide bonds; free amino acids do not give a positive result.

Summary

Amino acid: central carbon with amine group, carboxyl group, hydrogen and a variable R group; 20 kinds.
Peptide bond forms by condensation between –COOH of one amino acid and –NH₂ of the next; hydrolysis breaks it.
Primary: amino acid sequence (peptide bonds). Secondary: α-helix and β-pleated sheet (hydrogen bonds between backbone C=O and N–H).
Tertiary: specific 3-D shape held by hydrophobic interactions, hydrogen bonds, ionic bonds and disulfide bonds between R groups.
Quaternary: two or more polypeptides (sometimes with prosthetic groups), as in haemoglobin and collagen.
Denaturation: loss of 3-D shape (not primary structure) by heat or pH, causing loss of function.
Globular proteins: generally soluble, physiological roles. Fibrous proteins: generally insoluble, structural roles.

Practice questions

Question

Draw the general structure of an amino acid and label the amine group, carboxyl group and R group.
Name the reaction that forms a peptide bond, and the reaction that breaks it.
A protein has two polypeptide chains, of 21 and 30 amino acids. How many peptide bonds does it contain?
State the type of bond that holds the secondary structure of a protein in place, and the groups involved.
List the four types of interaction that maintain the tertiary structure of a protein.
Explain why globular proteins such as enzymes are usually soluble in water. (3 marks)
State two differences between globular and fibrous proteins.
Explain why a change in pH may change the shape of a protein. (3 marks)
Explain what is meant by quaternary structure, giving an example.
A scientist heats a solution of an enzyme to 80 °C for ten minutes, then cools it. The enzyme no longer works. A biuret test is still positive. Explain both observations. (5 marks)

Answers

Central C bonded to H, $-\text{NH}_2$ (amine group), $-\text{COOH}$ (carboxyl group) and R (R group/side chain).
Condensation; hydrolysis.
$(21 - 1) + (30 - 1) = 20 + 29 = 49$ . (This is insulin.)
Hydrogen bonds; between the $\text{C=O}$ group of one amino acid and the $\text{N–H}$ group of another in the polypeptide backbone.
Hydrophobic interactions; hydrogen bonds; ionic bonds; disulfide bonds (covalent).
Polypeptide folds so that hydrophobic R groups are on the inside, away from water; hydrophilic (polar or charged) R groups are on the outside; these form hydrogen bonds/interact with water molecules, so the protein dissolves.
Any two: globular are compact/spherical, fibrous long strands; globular generally soluble, fibrous insoluble; globular physiological/metabolic role, fibrous structural; globular have irregular amino acid sequences, fibrous often repetitive; globular more easily denatured.
Change in $\text{H}^+$ concentration alters the charge on R groups (ionisable groups gain or lose $\text{H}^+$ ); ionic bonds (and hydrogen bonds) between R groups break; the tertiary structure / 3-D shape changes.
Quaternary structure is the association of two or more polypeptide chains (sometimes with prosthetic groups) to form a functional protein, held by the same interactions as tertiary structure; e.g. haemoglobin (two α and two β chains plus haem groups) or collagen (three chains).
Heating increases kinetic energy, causing vibration that breaks hydrogen bonds, ionic bonds and hydrophobic interactions; the tertiary structure changes (denaturation) and the active site loses its specific shape, so substrate cannot bind; this is irreversible, so cooling does not restore activity. Peptide bonds are not broken (primary structure intact), and biuret reagent reacts with peptide bonds, so the biuret test is still positive.

Amino acids

The peptide bond

Four levels of protein structure

Primary structure

Secondary structure

Tertiary structure

Quaternary structure

Interactions that hold proteins in shape

Denaturation

Globular and fibrous proteins

Practice questions

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