Amino Acids, Peptides and the Levels of Protein Structure

Proteins carry out nearly every dynamic task a cell performs — catalysis, transport, signalling, structural support, immune recognition — and every one of these functions is dictated by a three-dimensional shape built, ultimately, from a one-dimensional sequence of twenty amino acids. This chapter opens the biochemistry block of the course by building that hierarchy from the ground up: the chemistry of a single amino acid, the covalent chemistry that links amino acids into chains, and the four distinct levels of structure — primary, secondary, tertiary and quaternary — through which a linear sequence folds into a functioning macromolecule.

Everything that follows in this block, from enzyme catalysis to the molecular machinery of the central dogma, assumes fluency with this chapter's vocabulary: the side chain ($\text{R}$ group) as the chemical identity of a residue, the peptide bond as the sole covalent link of the backbone, and the non-covalent forces — hydrogen bonds, hydrophobic interactions, van der Waals contacts, ionic bridges — that fold a chain into a shape precise enough to be useful. Treat the classification of the twenty side chains as material to memorise with structure firmly in mind, not as an isolated list.

1. The General Structure of an Amino Acid

With the exception of proline, all twenty standard amino acids share a common skeleton: a central alpha ($\alpha$) carbon bonded to four different groups — an amino group ($-\text{NH}_2$), a carboxyl group ($-\text{COOH}$), a hydrogen atom, and a variable side chain, denoted $\text{R}$, that gives each amino acid its distinct chemical identity.

Because the $\alpha$-carbon is bonded to four different substituents whenever $\text{R} \neq \text{H}$ (true for every amino acid except glycine, whose side chain is a second hydrogen atom), it is a stereocentre. Amino acids found in proteins are, with very rare exceptions, exclusively the L-enantiomer — a biosynthetic convention, not a thermodynamic necessity, since the D- and L-forms are equally stable in isolation.

Key distinction. "Amino acid" and "residue" are not strictly synonyms. A free amino acid in solution carries both a free $-\text{NH}_2$ and a free $-\text{COOH}$. Once incorporated into a peptide chain, one or both of these groups is consumed in forming a peptide bond, and the unit is more precisely called a residue.

2. Classification of the Twenty Side Chains

The twenty standard side chains are grouped, following the scheme used throughout Lehninger, by the chemical character they bring to a folded protein:

• Nonpolar, aliphatic: glycine, alanine, valine, leucine, isoleucine, methionine and proline. Proline is unique: its side chain cyclises back onto the amino nitrogen, creating a rigid secondary amine (not a primary amine) that constrains the backbone dihedral angle $\phi$ and frequently introduces a kink or a turn.
• Aromatic: phenylalanine, tyrosine and tryptophan. All three absorb ultraviolet light; tyrosine and tryptophan dominate a protein's absorbance at $280~\text{nm}$, the basis of a common concentration assay (anticipating the Beer–Lambert law developed later in this block).
• Polar, uncharged: serine, threonine, cysteine, asparagine and glutamine. Cysteine's thiol ($-\text{SH}$) is the one side chain capable of forming a covalent cross-link — a disulfide bond — with a second cysteine under oxidising conditions.
• Positively charged (basic): lysine, arginine and histidine. At physiological $\text{pH} \approx 7.4$, lysine's $\varepsilon$-amino group and arginine's guanidinium group are almost fully protonated; histidine's imidazole side chain, with a $\text{p}K_a$ near neutral pH, is the one side chain that can act as either an acid or a base under physiological conditions — the chemical basis of its frequent role at enzyme active sites (returned to in the next chapter).
• Negatively charged (acidic): aspartate and glutamate. Both carry a second carboxyl group on the side chain, fully ionised at physiological pH.

Hydropathy — a side chain's tendency to prefer an aqueous or a nonpolar environment — is the single property that does the most work in tertiary structure (Section 6): nonpolar and aromatic side chains are driven out of water and into a folded protein's interior, while charged and polar side chains remain solvent-exposed.

3. Acid–Base Behaviour: Zwitterions, $\text{p}K_a$ and the Isoelectric Point

Both the $\alpha$-amino and $\alpha$-carboxyl groups are ionisable, and under the conditions found in cells (and in most laboratory buffers), an amino acid exists as a zwitterion: a species carrying both a positive and a negative formal charge, with the carboxyl group deprotonated ($-\text{COO}^-$) and the amino group protonated ($-\text{NH}_3^+$), even though the net charge of the whole molecule is zero.

Glycine, the simplest amino acid, illustrates the full titration. Its carboxyl group has $\text{p}K_{a1} = 2.34$ and its amino group has $\text{p}K_{a2} = 9.60$ (standard values tabulated in Lehninger). Three ionic forms exist, interconverted by the Henderson–Hasselbalch equation as $\text{pH}$ is raised from strongly acidic to strongly basic:

The isoelectric point, $\text{pI}$, is the $\text{pH}$ at which the average net charge of the molecule is exactly zero. For a simple diprotic amino acid such as glycine, with no ionisable side chain between the two titrating groups, the isoelectric point lies midway between the two flanking $\text{p}K_a$ values:

Worked example. Find the isoelectric point of glycine, and determine its net charge at $\text{pH} = 7.0$.

Taking $\text{p}K_{a1} = 2.34$ and $\text{p}K_{a2} = 9.60$:

At $\text{pH} = 7.0$, a value well above $\text{p}K_{a1}$ and well below $\text{p}K_{a2}$, the carboxyl group is essentially fully deprotonated and the amino group essentially fully protonated: the molecule is overwhelmingly the zwitterion, net charge $0$. Since $\text{pH} = 7.0 > \text{pI} = 5.97$, glycine carries, on average, a very slightly negative charge — consistent with the general rule that a molecule above its $\text{pI}$ is net negative, and below its $\text{pI}$ is net positive.

For an amino acid with an ionisable side chain (e.g. aspartate, side-chain $\text{p}K_a \approx 3.86$; histidine, side-chain $\text{p}K_a \approx 6.00$), three $\text{p}K_a$ values exist rather than two, and the isoelectric point is the average of the two $\text{p}K_a$ values that flank the zwitterionic (net-zero) species specifically — not simply the two smallest or two largest. This distinction is the basis of electrophoresis and isoelectric focusing, techniques that separate proteins by the $\text{pH}$ at which they carry no net charge and therefore stop migrating in an electric field.

Definition. The isoelectric point, $\text{pI}$, is the pH at which a molecule's average net charge is zero. It is a property of the molecule's ionisable groups alone, found by averaging the two $\text{p}K_a$ values that bracket the zero-charge species on a titration curve.

4. The Peptide Bond

Amino acids are joined into chains by a peptide bond: an amide linkage formed by the condensation of the $\alpha$-carboxyl group of one amino acid with the $\alpha$-amino group of the next, releasing one molecule of water.

The resulting $-\text{CO-NH}-$ linkage is planar and, critically, exhibits partial double-bond character: resonance between the carbonyl oxygen and the amide nitrogen restricts rotation about the $\text{C-N}$ bond, so the six atoms of the peptide unit ($\text{C}_\alpha$, $\text{C}$, $\text{O}$, $\text{N}$, $\text{H}$, $\text{C}_\alpha$ of the next residue) lie in a single rigid plane, almost always in the lower-energy trans configuration. This rigidity is what makes the backbone's conformational freedom reducible to just two rotatable dihedral angles per residue, $\phi$ (phi, around $\text{N-C}_\alpha$) and $\psi$ (psi, around $\text{C}_\alpha\text{-C}$) — the variables plotted on the Ramachandran plot of Section 6.

A chain of amino acids linked this way is a polypeptide; by convention it is written and numbered from its N-terminus (the residue retaining a free $\alpha$-amino group) to its C-terminus (the residue retaining a free $\alpha$-carboxyl group). Short chains (conventionally fewer than about 20–50 residues) are called peptides; longer chains with a defined, stable fold are proteins.

5. Primary Structure

The primary structure of a protein is simply its covalent structure: the linear sequence of residues from N-terminus to C-terminus, together with the location of any disulfide bonds. It is the one level of structure specified directly by the gene (via the genetic code developed in the next chapters of this block) and is, in Anfinsen's classic principle, sufficient on its own to determine every higher level of structure for most single-domain proteins — the sequence specifies the fold.

Primary structure is conventionally written using either the three-letter or single-letter amino acid code, e.g. Gly-Ser-Ala or GSA, always read N-terminus to C-terminus. Classical sequence determination (Edman degradation, which sequentially removes and identifies one N-terminal residue at a time) has been largely superseded by mass spectrometry, covered later in this block, but the N-to-C reading convention established here persists throughout.

6. Secondary Structure: the $\alpha$-Helix and the $\beta$-Sheet

Secondary structure describes regular, locally repeating conformations of the backbone, stabilised entirely by hydrogen bonds between backbone carbonyl oxygens and backbone amide hydrogens — side chains play no direct role in forming these bonds, though they strongly influence which secondary structures a given sequence favours.

6.1 The $\alpha$-helix

The $\alpha$-helix is a right-handed coil in which the backbone hydrogen-bonds each carbonyl oxygen of residue $n$ to the amide hydrogen of residue $n+4$, producing a hydrogen-bonding pattern that runs parallel to the helix axis. Its geometry is fixed and reproducible across proteins:

• Rise per residue: $1.5~\text{\AA}$ ($0.15~\text{nm}$) along the helix axis.
• Residues per turn: $3.6$.
• Pitch (rise per complete turn): $3.6 \times 1.5~\text{\AA} \approx 5.4~\text{\AA}$.

Worked example. A protein segment forms a perfect $\alpha$-helix of $22$ residues. Estimate its length along the helix axis.

Using the rise per residue, $1.5~\text{\AA}$:

Proline disrupts the $\alpha$-helix for two independent reasons: its ring fixes $\phi$ at a value incompatible with the helical conformation, and its secondary-amine nitrogen has no amide hydrogen left to donate to the $n$-to-$n{+}4$ hydrogen bond. Glycine, by contrast, destabilises regular helices for the opposite reason — its tiny side chain gives the backbone so much conformational freedom that it is entropically costly to confine it to one geometry, so glycine is instead enriched in tight turns.

6.2 The $\beta$-sheet

The $\beta$-sheet arranges the backbone into near-fully-extended strands, hydrogen-bonded side by side (rather than within a single stretch of chain) to form a pleated sheet. Adjacent strands can run in the same N-to-C direction (parallel $\beta$-sheet) or in opposite directions (antiparallel $\beta$-sheet); the antiparallel arrangement allows straight, evenly spaced hydrogen bonds and is correspondingly the more stable of the two.

6.3 The Ramachandran plot

Plotting every residue's $(\phi, \psi)$ pair on a Ramachandran plot reveals that most combinations are sterically forbidden by backbone-atom clashes; only a few regions are energetically accessible, corresponding closely to the $\alpha$-helix and $\beta$-sheet conformations described above. Glycine, lacking a side chain, accesses a much larger region of the plot than any other residue, which is exactly why it is tolerated at tight turns no other residue could adopt.

7. Tertiary Structure

Tertiary structure is the complete three-dimensional fold of a single polypeptide chain — the overall arrangement in space of every secondary-structure element and every side chain. Four non-covalent interactions, plus one covalent cross-link, stabilise it:

• Hydrophobic interactions. The dominant driving force for folding: nonpolar side chains cluster into a solvent-excluded core, while polar and charged side chains remain surface-exposed. This "hydrophobic collapse" is favoured thermodynamically because it releases ordered water molecules from around nonpolar surfaces, increasing the entropy of the solvent.
• Hydrogen bonds between side chains, and between side chains and the backbone.
• Ionic interactions (salt bridges) between oppositely charged side chains, e.g. a lysine $\varepsilon$-amino group and a glutamate carboxylate.
• Van der Waals interactions, individually weak but numerous enough, in a tightly packed hydrophobic core, to contribute substantially to overall stability.
• Disulfide bonds, the one covalent cross-link, formed by oxidation of two cysteine thiols: $2\ \text{R-SH} \rightarrow \text{R-S-S-R} + 2\text{H}^+ + 2e^-$. Disulfides are common in secreted extracellular proteins (the extracellular environment is oxidising) and rare in the cytosol (which is reducing).

Key distinction. Only the disulfide bond is covalent. Every other stabilising interaction listed above is individually weak and non-covalent; it is their large number, not their individual strength, that makes a folded protein thermodynamically stable relative to the unfolded chain.

Folding in the cell is assisted by molecular chaperones (e.g. the Hsp70 family, and the GroEL/GroES chaperonin complex), which bind exposed hydrophobic surfaces of a nascent or stress-denatured chain, preventing aggregation and giving the chain repeated opportunities to find its correct fold — the chaperone does not specify the fold itself, which remains encoded in the sequence (Section 5).

Denaturation is the loss of tertiary (and secondary) structure without breaking peptide bonds, caused by heat, extremes of $\text{pH}$, chaotropic agents (urea, guanidinium chloride, which disrupt the hydrophobic effect), detergents such as SDS (which coat and solubilise the hydrophobic core), or reducing agents such as dithiothreitol (DTT) or $\beta$-mercaptoethanol (which cleave disulfide bonds). Some small, single-domain proteins refold spontaneously and completely once the denaturant is removed — the experimental basis of Anfinsen's principle that sequence alone determines the native fold.

8. Quaternary Structure

Many functional proteins are assemblies of more than one polypeptide chain, each called a subunit. Quaternary structure describes the number and spatial arrangement of these subunits, held together by the same non-covalent forces (and occasionally interchain disulfide bonds) that stabilise tertiary structure within a single chain.

Haemoglobin is the standard illustration: a tetramer of two $\alpha$-globin and two $\beta$-globin subunits ($\alpha_2\beta_2$), each individually folded into a tertiary structure closely related to myoglobin, assembled into a single oxygen-carrying quaternary unit. A protein consisting of a single polypeptide chain, by definition, has no quaternary structure at all — the level simply does not apply.

9. Common Exam Pitfalls

• Confusing "amino acid" with "residue." Once incorporated into a chain, a unit has lost a water molecule relative to the free amino acid; always state clearly whether a calculation concerns free amino acids or residues within a peptide.
• Computing the isoelectric point as a simple average of the wrong two $\text{p}K_a$ values. For an amino acid with three ionisable groups, $\text{pI}$ is the average of the two $\text{p}K_a$ values that flank the net-zero-charge (zwitterionic) species on the titration curve — not automatically the smallest two or the largest two.
• Treating the peptide bond as freely rotatable. Resonance gives it partial double-bond character, fixing the six peptide-unit atoms in a single rigid plane (almost always trans); only the $\phi$ and $\psi$ dihedral angles are free to rotate.
• Assuming side chains form the hydrogen bonds of secondary structure. The $\alpha$-helix and $\beta$-sheet are defined entirely by backbone-to-backbone hydrogen bonds; side chains influence which structure forms but do not participate in these particular bonds.
• Forgetting that denaturing agents do not break peptide bonds. Heat, pH, urea, SDS and reducing agents disrupt non-covalent interactions (and, for reducing agents, disulfide bonds specifically) — the primary sequence survives denaturation intact.
• Assigning quaternary structure to a single-chain protein. A monomeric protein has primary, secondary and tertiary structure only; quaternary structure requires at least two independently folded polypeptide chains.

Summary

• An amino acid has a central $\alpha$-carbon bearing an amino group, a carboxyl group, a hydrogen, and a variable side chain $\text{R}$; the twenty side chains are classed as nonpolar aliphatic, aromatic, polar uncharged, positively charged, or negatively charged.
• Amino acids exist as zwitterions near neutral pH; the isoelectric point, $\text{pI} = \dfrac{\text{p}K_{a1}+\text{p}K_{a2}}{2}$ for a simple amino acid, is the pH of zero average net charge.
• The peptide bond is a planar, resonance-stabilised amide linkage formed with loss of water; chains are read N-terminus to C-terminus.
• Primary structure is the covalent sequence; secondary structure ($\alpha$-helix, rise $1.5~\text{\AA}$/residue, $3.6$ residues/turn; $\beta$-sheet, parallel or antiparallel) arises from backbone hydrogen bonds alone.
• Tertiary structure is stabilised chiefly by the hydrophobic effect, assisted by hydrogen bonds, salt bridges, van der Waals contacts and covalent disulfide bonds; chaperones assist folding without dictating the fold.
• Quaternary structure describes the assembly of multiple folded subunits (e.g. haemoglobin, $\alpha_2\beta_2$) and does not apply to single-chain proteins.
• The next chapter builds directly on tertiary structure to explain how a folded protein — an enzyme — achieves catalysis, culminating in Michaelis–Menten kinetics.