Peptide bonds and the levels of protein structure

Joining two amino acids

The carboxyl group of one amino acid and the amino group of another can react. A molecule of water is lost, and a new bond forms between the carbonyl carbon and the nitrogen:

Two amino acids, $\text{H}_{2}\text{N}\text{-}\text{CH}(\text{R}_{1})\text{-}\text{COOH}$ and $\text{H}_{2}\text{N}\text{-}\text{CH}(\text{R}_{2})\text{-}\text{COOH}$, react. The product is a dipeptide, with the two parts $\text{H}_{2}\text{N}\text{-}\text{CH}(\text{R}_{1})\text{-}\text{CO}\text{-}$ and $\text{-}\text{NH}\text{-}\text{CH}(\text{R}_{2})\text{-}\text{COOH}$ joined at the new bond, and one molecule of water.

This C to N bond is a peptide bond. It is an amide. Two amino acids give a dipeptide, three give a tripeptide, and a chain of many is a polypeptide. A protein is one polypeptide chain, or several, folded into a fixed shape.

Each amino acid in the chain is a residue. A chain has one free amino group, the N-terminal end, and one free carboxyl group, the C-terminal end. We write the sequence from the N end to the C end. Gly-Ala means that glycine has the free amino group, and alanine has the free carboxyl group. Ala-Gly is a different dipeptide.

A chain of $n$ residues has $n - 1$ peptide bonds. It was made by releasing $n - 1$ molecules of water. Complete hydrolysis puts the water back. Boiling in 6 M hydrochloric acid for about 24 hours does this, and it gives the free amino acids.

Why the peptide bond is flat

The lone pair on the nitrogen atom spreads over the C to N bond, so the bond is neither a pure single bond nor a pure double bond. This is resonance. The C to N bond in a peptide is 0.133 nm long. A normal C to N single bond is 0.145 nm, and a C to N double bond is 0.125 nm. So the peptide bond is a partial double bond.

Because of this, the six atoms of the group (the two α carbons, the carbonyl carbon and oxygen, and the nitrogen and its hydrogen) lie in one plane. The two α carbons are on opposite sides, which is the trans arrangement. Rotation about the C to N bond is not free. The chain can turn only about the bonds on either side of each α carbon.

Counting the sequences

A protein is defined by its order of residues. With 20 amino acids and $n$ places, there are $20^n$ possible sequences:

• A dipeptide: $20^2 = 400$.
• A tripeptide: $20^3 = 8000$.
• A chain of 100 residues: $20^{100}$, which is about $10^{130}$.

If a peptide must use three particular amino acids once each, there are $3! = 6$ orders. If some residues repeat, divide by the factorial of each repeat. Gly, Gly, Ala can be arranged in $3!/2! = 3$ ways.

The four levels of structure

1. Primary structure is the sequence of residues, held by peptide bonds (covalent bonds). It decides every other level.
2. Secondary structure is the regular pattern of the backbone, held by hydrogen bonds between the C=O group of one peptide and the N-H group of another.
3. Tertiary structure is the way the whole chain folds in three dimensions.
4. Quaternary structure is the way several folded chains pack together.

The α-helix. The chain coils like a spring. It is right-handed, with 3.6 residues in each turn. Each residue adds 0.15 nm to the length, so one turn is 0.54 nm. The C=O of residue $i$ forms a hydrogen bond with the N-H of residue $i + 4$. The side chains point outward. Keratin, in hair and nails, is rich in α-helix.

The β-pleated sheet. The chains are stretched, with about 0.33 nm per residue, and lie side by side. Hydrogen bonds join the neighbouring strands, and the sheet is pleated like a folded paper. The strands can run in the same direction (parallel) or in opposite directions (antiparallel). Silk fibroin is rich in β-sheet.

Tertiary structure. Five kinds of interaction hold a folded chain:

• Disulphide bridges, $\text{-}\text{S}\text{-}\text{S}\text{-}$, which are covalent bonds between two cysteine residues.
• Hydrogen bonds between side chains.
• Ionic attractions, also called salt bridges, between $\text{-}\text{COO}^{-}$ and $\text{-}\text{NH}_{3}^{+}$ side chains.
• Hydrophobic interactions: the non-polar side chains pack together inside the protein, away from the water.
• Van der Waals forces between close atoms.

Quaternary structure. Haemoglobin has four chains, two α and two β. Each chain has 141 or 146 residues, so the whole molecule has $2 \times 141 + 2 \times 146 = 574$ residues. Insulin has two chains, A (21 residues) and B (30 residues), joined by two disulphide bridges, so it has 51 residues.

Proteins fall into two broad classes. Fibrous proteins, such as keratin, collagen and silk, are long and thread-like, and they do not dissolve in water. Globular proteins, such as albumin, insulin, haemoglobin and the enzymes, fold into a compact ball and dissolve in water.

Slide the share of the helix from 0% to 100%. The length of the chain falls to less than half, but the number of peptide bonds does not change. Folding changes the shape and not the primary structure.

Practise with new numbers

Try it in Python

The program works out the number of peptide bonds and the molar mass of four peptides. A letter stands for an amino acid: G is glycine, A alanine, S serine, V valine, L leucine, F phenylalanine and D aspartic acid.

Two colour tests

• Biuret test. A peptide with at least two peptide bonds gives a violet colour with copper(II) sulphate in alkali. A dipeptide has only one bond and gives no colour. The test shows the presence of protein.
• Xanthoproteic test. Concentrated nitric acid turns the benzene rings of tyrosine, phenylalanine and tryptophan yellow, and then orange with alkali.

Common mistakes

• Counting the peptide bonds as the number of residues. A chain of $n$ residues has $n - 1$ bonds.
• Writing Gly-Ala and Ala-Gly as the same dipeptide. They have different N and C ends.
• Thinking the peptide bond rotates freely. It is a partial double bond, flat and trans.
• Mixing up primary and secondary structure. The primary structure is only the sequence.

Remember these

• The peptide bond is an amide, made by losing one water. A chain of $n$ residues has $n - 1$ bonds.
• The bond is flat, trans and about 0.133 nm long, a partial double bond.
• Primary: sequence. Secondary: α-helix (3.6 residues per turn, 0.15 nm per residue) and β-sheet. Tertiary: the whole fold. Quaternary: several chains.
• Fibrous proteins are insoluble. Globular proteins dissolve in water.

Check yourself

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

1. How many tripeptides can be made from the three amino acids Gly, Ala and Ser, using each once?

2. A protein has 300 residues in one chain. How many peptide bonds does it have, and how many molecules of water must be added to hydrolyse it completely?

3. Which level of structure is broken when a disulphide bridge between two cysteine residues is cut?

4. Why is the peptide bond flat?

References

• Nelson and Cox, Lehninger Principles of Biochemistry, 8th edition, Chapter 3 (peptide bond) and Chapter 4 (levels of protein structure).
• NCERT Chemistry for Class 12, Part II, the chapter Biomolecules (proteins and their structure).
• OpenStax, Organic Chemistry, Chapter 26 (Biomolecules: Amino Acids, Peptides and Proteins).