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T1

Proteins

AQA spec ref: 3.1.4 - Proteins

Proteins are the most structurally and functionally diverse molecules in biology. They are polymers of amino acids, and their enormous range of functions - enzymes, structural proteins, hormones, antibodies, transport proteins, receptors - arises entirely from the variety of shapes that polypeptide chains can fold into. Understanding the four levels of protein structure is the foundation for understanding almost everything else in biology.

Amino Acid Structure

All amino acids share the same basic structure centred on a central (alpha) carbon to which four groups are attached:

  1. An amino group (-NH₂) - basic
  2. A carboxyl group (-COOH) - acidic
  3. A hydrogen atom (-H)
  4. A variable R group (side chain) - this is what differs between amino acids

There are 20 different naturally occurring amino acids, each with a different R group. The R group determines the amino acid's chemical properties:

  • Non-polar/hydrophobic R groups (e.g. alanine, valine, leucine) - tend to be found on the inside of a protein, away from the aqueous environment
  • Polar/hydrophilic R groups (e.g. serine, threonine) - often on the outside, interact with water
  • Charged R groups (e.g. glutamic acid = negative; lysine = positive) - form ionic bonds or interact with other charged groups
  • R groups with -SH (cysteine) - form disulfide bonds with other cysteine residues

Peptide Bonds

Amino acids are joined by condensation reactions between the carboxyl group (-COOH) of one amino acid and the amino group (-NH₂) of the next. A water molecule is released and a peptide bond (-CO-NH-) is formed.

A chain of amino acids joined by peptide bonds is a polypeptide. A protein may consist of one or more polypeptide chains. The peptide bond is a covalent bond - stronger than the non-covalent interactions that determine the protein's shape. Polypeptides are broken down by hydrolysis (adding water, catalysed by proteases).

The chain of a polypeptide has:

  • N-terminus (free -NH₂ group) at one end
  • C-terminus (free -COOH group) at the other
  • Polypeptides are conventionally written N→C

Primary Structure

The primary structure is the specific sequence of amino acids in a polypeptide chain, determined by the DNA sequence of the gene. It is the unique "identity" of the protein - two polypeptides with different primary structures will always have different shapes and functions. A single amino acid change (due to a missense mutation) can profoundly alter protein function (e.g. sickle cell haemoglobin).

Secondary Structure

The polypeptide chain folds into regular, repeating local structures stabilised by hydrogen bonds between the oxygen of the carbonyl group (-C=O) and the hydrogen of the amino group (-N-H) of the backbone (not R groups). Two main types:

Alpha helix (α-helix) - the polypeptide chain coils into a right-handed helix. Hydrogen bonds form between every 4th amino acid along the chain. The R groups project outward. Alpha helices are found in structural proteins (e.g. keratin - which is almost entirely alpha helices coiled around each other) and in globular proteins (as sections embedded in membranes, since alpha helices with hydrophobic R groups can span the lipid bilayer).

Beta-pleated sheet (β-sheet) - the polypeptide chain zigzags back and forth, with hydrogen bonds forming between adjacent parallel or antiparallel strands. The R groups project above and below the sheet. Beta-pleated sheets are found in silk fibroin (giving silk its flexible yet strong structure) and in many enzyme active sites.

Tertiary Structure

The tertiary structure is the overall three-dimensional folding of a single polypeptide chain. It is stabilised by interactions between R groups:

  1. Disulfide bonds ( - S - S - ) - covalent bonds between the -SH groups of two cysteine residues. These are the strongest interactions maintaining tertiary structure (covalent > non-covalent). Formed by oxidation of two -SH groups.
  1. Ionic bonds - electrostatic attractions between positively and negatively charged R groups (e.g. between lysine -NH₃⁺ and glutamic acid -COO⁻). Weakened by extremes of pH, which alter the charge on R groups.
  1. Hydrogen bonds - between polar R groups ( - OH, - NH₂, - COOH) that are not involved in secondary structure. Individually weak but collectively important. Disrupted by high temperature (denaturation).
  1. Hydrophobic interactions - non-polar R groups cluster together on the inside of the protein, away from the aqueous environment. This is entropically driven (releasing ordered water molecules from around hydrophobic groups). Not really a bond, but a driving force for folding.

The tertiary structure determines the protein's 3D shape and therefore its function - the shape of an enzyme's active site, the shape of an antibody's antigen-binding site, the shape of a receptor's binding pocket. This is why denaturation (unfolding of tertiary structure) destroys function.

Quaternary Structure

Quaternary structure is the association of two or more polypeptide chains (subunits) into a single functional protein. The subunits are held together by the same interactions that maintain tertiary structure (hydrogen bonds, ionic bonds, hydrophobic interactions, and sometimes disulfide bonds between chains).

Examples:

  • Haemoglobin - 4 subunits: 2α + 2β polypeptide chains, each associated with a haem group (non-polypeptide prosthetic group). The quaternary structure enables cooperativity in O₂ binding.
  • Collagen - 3 polypeptide chains wound around each other in a triple helix, with hydrogen bonds and covalent cross-links between chains.
  • Insulin - 2 chains (A and B) linked by disulfide bonds.

Prosthetic groups are non-polypeptide components permanently associated with a protein. Examples: haem group in haemoglobin (iron-containing), retinal in rhodopsin, carbohydrate chains on glycoproteins.

Fibrous Proteins

Fibrous proteins are structural - they are insoluble in water, have regular, repetitive primary structures, and form long fibres or sheets. Their repeating sequences fold into regular secondary structures that pack together for strength.

Collagen - the most abundant protein in mammals (~30% of all protein). Found in tendons, ligaments, skin, cartilage, bone, and blood vessels.

  • Three polypeptide chains (each an alpha chain) wound around each other in a right-handed triple helix - this is the quaternary structure.
  • Each chain has a repeating triplet sequence Gly-X-Y (glycine every third residue). Glycine is the smallest amino acid - essential because only glycine can fit into the centre of the triple helix.
  • The chains are held together by hydrogen bonds within and between chains.
  • Collagen molecules are then cross-linked to each other by covalent bonds to form collagen fibrils, which bundle into collagen fibres with enormous tensile strength.
  • Function: provides tensile strength - resists pulling forces.

Keratin - found in hair, nails, feathers, scales, horns. Consists of alpha helices coiled around each other (coiled coils) stabilised by disulfide bonds. Many disulfide bonds = brittle keratin (nails, horn); fewer = flexible keratin (hair).

Elastin - found in skin, blood vessels, lung tissue. Crosslinked polypeptide chains that can be stretched and recoil elastically.

Globular Proteins

Globular proteins are compact, roughly spherical, generally water-soluble (hydrophilic R groups on the surface, hydrophobic R groups in the interior), and functionally active - they include enzymes, hormones, antibodies, receptors, and transport proteins.

Haemoglobin - oxygen transport in blood. Four subunits, each with an iron-containing haem group that binds one O₂ molecule. See Mass Transport in Animals for oxygen dissociation and the Bohr effect.

Enzymes - globular proteins with a specific active site shaped to bind a complementary substrate. The active site is formed by the tertiary (and sometimes quaternary) structure. Denaturation = loss of active site shape = loss of function. See dedicated enzyme notes if they exist.

Insulin - pancreatic hormone; two chains linked by disulfide bonds. See Hormonal Control.

Biuret Test for Proteins

The Biuret test detects peptide bonds (and therefore polypeptides/proteins):

  1. Add a few drops of sodium hydroxide (NaOH) to make the solution alkaline
  2. Add a few drops of copper sulfate solution (CuSO₄) (dilute)
  3. Mix and observe colour
  • Positive result: a purple/lilac colour develops - the Cu²⁺ ions coordinate with the nitrogen atoms in the peptide bonds under alkaline conditions.
  • Negative result: the solution remains blue (the colour of CuSO₄ alone).

The intensity of the purple colour is proportional to the concentration of protein.

Summary

  • Amino acid: central C with -NH₂, -COOH, -H, and R group. 20 different amino acids (different R groups).
  • Peptide bonds: formed by condensation between -COOH and -NH₂; broken by hydrolysis.
  • Primary: amino acid sequence (coded by DNA)
  • Secondary: alpha helix (H-bonds along backbone, coiled) or beta-pleated sheet (H-bonds between strands)
  • Tertiary: 3D folding via R group interactions - disulfide bonds (covalent), ionic bonds, H-bonds, hydrophobic interactions
  • Quaternary: two or more polypeptide chains (subunits) together; e.g. haemoglobin (4 subunits), collagen (3 chains)
  • Fibrous: insoluble, structural (collagen, keratin, elastin)
  • Globular: soluble, metabolically active (enzymes, hormones, haemoglobin, antibodies)
  • Biuret test: NaOH+CuSO₄→purple=protein present

AQA Exam Tips

  • Primary structure determines everything else: the sequence of amino acids determines which R groups are present, which determines what interactions can form, which determines the tertiary shape, which determines function. This chain of causation is fundamental.
  • Denaturation: irreversible unfolding of tertiary and quaternary structure by heat (vibration breaks H-bonds and hydrophobic interactions) or extremes of pH (alters ionisation of R groups, disrupts ionic bonds and H-bonds). Peptide bonds (primary structure) are NOT broken by denaturation - use a protease for that.
  • Collagen structure: AQA loves the Gly-X-Y repeat and the triple helix. State that glycine must be at every third position because it is the only amino acid small enough to fit in the centre of the triple helix.
  • Disulfide bonds are covalent: the other forces maintaining tertiary structure are non-covalent (H-bonds, ionic, hydrophobic). Disulfide bonds are the strongest because they are covalent.
  • Biuret test: state sodium hydroxide is added first to make the solution alkaline, THEN copper sulfate. Purple colour. Do not confuse with Benedict's (for reducing sugars) or iodine (for starch).
  • Enzymes: the active site has a shape complementary to the substrate. The shape arises from tertiary structure. Any factor that changes tertiary structure (temperature above optimum, wrong pH) will change the active site shape and reduce/eliminate enzyme activity.