Lecture 1: proteins, pka, amino acids

Structure of a typical amino acid

• There are 20 common amino acids found in proteins; they differ according to their side chains (R group).

• The amino group and the carboxyl group are ionizable (i.e., can gain or lose H+).

Amino acid chirality

• Amino acids are non-superimposable due to 1 chiral centre.

• A chiral carbon is bonded to 4 DIFFERENT atoms or groups.

Properties of amino acids

• Properties are conferred by the R group.

• They are chiral (all except glycine, whose R group is H, so there are only 3 different groups bonded to the central C atom).

• L-amino acids and D-amino acids are mirror images (enantiomers) and cannot be superimposed.

• Only L-amino acids are found in proteins in cells; D-amino acids are found in the peptidoglycan cell walls of some bacteria, but not in bacterial proteins.

• D/L relate to configuration relative to (+) glyceraldehyde; all L-amino acids have the same steric configuration as L-glyceraldehyde.

Enantiomers

• Enantiomers are 2 stereoisomers that are non-superimposable mirror images of each other.

• They have the same physical and chemical properties but rotate plane-polarized light in opposite directions.

• A mixture of equal concentrations of 2 enantiomers is called a racemic mixture.

• The specific rotation is zero for a racemate because the rotations cancel out.

• Relevance to drugs: only one enantiomer may have activity or one may be toxic.

Acidic R Groups

• Examples include L-glutamic acid (Glu or E).

• Ionizable features shown in structural drawings include the ability to lose protons from functional groups.

Neutral-Nonpolar R Groups

• R groups are hydrocarbon; overall neutral and nonpolar.

• Includes glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), phenylalanine (Phe, F), methionine (Met, M), proline (Pro, P), cysteine (Cys, C) with a nonpolar thiol group.

• Cysteine’s -SH group is nonpolar but can form weak H bonds with O and N; can form disulfide bonds to form cystine. There are 2 sulfur-containing amino acids, cysteine and methionine, but only cysteine can form disulfide bonds.

Neutral-Polar R Groups

• R groups can hydrogen bond and are hydrophilic (water-loving).

• Include serine (Ser, S), threonine (Thr, T), glutamine (Gln, Q), asparagine (Asn, N).

• Serine and threonine have OH groups in their R groups; glutamine and asparagine are amides of glutamic and aspartic acid.

Acidic amino acids

• There are 2 acidic amino acids: glutamic acid (Glu, E) and aspartic acid (Asp, D).

• When ionized (COO−), they are called glutamate and aspartate.

• Glutamic acid: both COOH groups can ionize, giving glutamate and aspartate, respectively.

• Note: the drawings often show non-ionized forms; at physiological pH, both COOH and NH2 groups are ionized.

Basic amino acids

• There are 3 basic amino acids: arginine (Arg, R), lysine (Lys, K), and histidine (His, H).

• Arginine can be protonated at the guanidino group; lysine’s R group NH2 can be protonated to NH3+; histidine’s imidazole ring can be protonated gaining H+.

Proline

• The R group of proline is bonded to the amino nitrogen, so proline is technically an imino acid, not a standard amino acid.

• Nevertheless, it is commonly found in proteins.

Hydrophobic vs hydrophilic classification

• Two broad classes of amino acids based on R-group:

  • Hydrophobic: repelled by water; mostly interior of proteins; do not ionize or form hydrogen bonds.

  • Hydrophilic: interact with aqueous environment; often have -OH groups; frequently form hydrogen bonds; mostly on protein surfaces or in active sites of enzymes.

Titration and isoelectric point (pI)

• The predominant ionic form of an amino acid in solution depends on pH.

• At low pH, alanine carries a net positive charge; as pH increases, the -COOH group ionizes, leading to a zwitterion with no net charge; at high pH, the -NH3+ group loses a proton to become -NH2, giving a net negative charge.

• The pI is the pH at which the net charge is zero.

• R group for alanine: -CH3

Henderson-Hasselbalch equation

• For a weak acid, HA ⇌ H+ + A−

• Ka = [H+][A−]/[HA]

• pKa = -log Ka

• The relationship between pH and pKa is described by the Henderson–Hasselbalch equation:
  $\text{pH} = \text{p}K_a + \log\frac{[\text{A}^-]}{[\text{HA}]}$

• This enables prediction of the ratio of charged to uncharged forms at a given pH, given pKa.

Why Henderson–Hasselbalch is important

• Buffer calculations (e.g., blood acid-base status, buffer preparation in labs).

• Calculation of ionized/unionized concentrations of chemicals, including drugs.

• See examples and applications in pharmacology resources.

Calculating isoelectric point (pI)

• For alanine: pKa(COOH) ≈ 2.3; pKa(NH3+) ≈ 9.7.

• pI(alanine) = (pKa1 + pKa2) / 2 = (2.3 + 9.7) / 2 = 6.0.

• For glutamate or aspartate, the pI is halfway between the two carboxyl group pKa values.

• For lysine and arginine (basic amino acids), the pI is between the pKa values of the two amino groups.

pKa values for ionizable groups

• Neutral amino acids: 2 pKa values (for the α-carboxyl and α-amino groups).

• Acidic or basic amino acids: 3 pKa values because the R group ionizes.

• Approximate values:

  • pKa for the COOH of R groups in aspartate/glutamate ≈ 4

  • pKa for histidine R group ≈ 6–7

  • pKa for lysine R group ≈ 10

  • pKa for arginine R group ≈ 12

Useful references

• See LibreTexts for amino acids/proteins: https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3ABiochemistryFreeForAll(AhernRajagopalandTan)/02%3AStructureandFunction/202%3AStructureFunction-AminoAcids

• Henderson–Hasselbalch video and related buffers resources: Khan Academy

Peptide bonds

• Peptide bonds are rigid due to resonance stabilization, and are planar (flat) and resistant to hydrolysis.

• Formation is a condensation reaction; involves N-terminal and C-terminal ends of the polypeptide.

Levels of protein structure

• Primary structure: sequence of amino acids in a protein or peptide (the linked amino acids are residues).

  • The primary structure alone tells us nothing about 3D arrangement.

  • Convention: the first amino acid is the N-terminus.

• Secondary structure: includes α-helix and β-sheet (two main types).

The α-helix

• Held together by hydrogen bonds between the C=O group of one amino acid and the N–H group of another, four residues away (i to i+4).

• R groups protrude; not involved in maintaining the helical hydrogen-bond network.

• Found in almost all proteins; rigid, right-handed structure.

• Approximately 3.6 amino acids per turn.

• Proline tends to disrupt helices (ring restricts rotation); glycine is too small/flexible to stabilize an α-helix.

β-sheet

• Form when two or more segments line up side-by-side.

• Stabilized by hydrogen bonds between N–H and C=O groups of adjacent chains.

• Can be parallel or antiparallel (running in same or opposite directions).

• Many globular proteins contain mixtures of α-helix and β-sheet; some proteins (e.g., silk fibroin) are β-sheet–rich; others (e.g., myosin) are mainly α-helix.

Collagen triple helix

• Collagen is the most abundant protein in the body; major component of connective tissue.

• Composed of a triple helix, tightly wound.

• Every third amino acid is proline; contains hydroxyproline in addition to proline.

Tertiary structure

• The 3D conformation adopted by proteins when they fold.

• Folding brings distant residues in sequence into close spatial proximity, creating a compact structure.

• Large globular proteins are often composed of domains (structurally independent parts with separate functions).

• Example: hexokinase 3D space-filled model (enzyme that catalyses the first step in glycolysis).

Quaternary structure

• Some proteins consist of several polypeptide chains; subunits may be identical or different.

• If identical, proteins are oligomers.

• Quaternary structure can be important for function (e.g., cooperative ligand binding).

• Subunits held together by hydrophobic interactions, salt bridges, hydrogen bonds, and disulfide bonds.

• Classic example: haemoglobin (tetramer).

Prosthetic groups and non-protein components in quaternary structure

• Some quaternary structures involve binding to non-protein groups (prosthetic groups).

• Examples:

  • Haem in haemoglobin (binds Fe2+, which binds O2).

  • Sugars in glycoproteins (N- and O-linked glycosylation) added in ER and Golgi.

  • Coenzymes permanently linked to enzymes (e.g., pyridoxal phosphate in transaminase enzymes).

Protein stability and interactions (summary)

• Stabilizing forces in protein structure include:

  • Hydrophobic interactions (as proteins fold, hydrophobic R groups cluster away from water).

  • Hydrogen bonds (between R groups, and between R groups and the backbone).

  • Electrostatic interactions (salt bridges).

  • Weak van der Waals interactions.

  • Covalent bonds (disulfide bonds between cysteines; present in many extracellular proteins).

  • Posttranslational modifications (e.g., phosphorylation, glycosylation) can form covalent attachments.

Protein denaturation

• Denaturation disrupts protein structure and often causes loss of activity.

• Causes include:

  • Extremes of pH.

  • Organic solvents (some solvents disrupt hydrogen bonding, others disrupt hydrophobic interactions).

  • Detergents (e.g., SDS) disrupt hydrophobic interactions and unfold the molecule.

  • Reducing agents (e.g., β-mercaptoethanol) reduce disulfide bonds.

  • High temperature – hydrogen bonds are disrupted.

  • Heavy metals and high salt concentrations.

Note on terminology and resources

• Proline is an imino acid due to its ring-linked amino nitrogen.

• Cysteine can form disulfide bonds; two cysteines linked form cystine.

• Aromatic amino acids (phenylalanine, tyrosine, tryptophan) absorb ultraviolet light at 280 nm and are used in protein concentration measurements by spectrophotometry.

• Useful reference for amino acids/proteins: LibreTexts (see page link above).

• General concept: D- and L- nomenclature relates to glyceraldehyde configuration; only L-amino acids are incorporated into proteins in most organisms.

Quick reference values (useful anchors)

• Neutral amino acids: typically have 2 pKa values (α-COOH and α-NH3+).

• Acidic or basic amino acids: typically have 3 pKa values because the R group ionizes.

• Common approximate pKa values:

  • COOH of Asp/Glu side chains: ~4

  • Imidazole (His) R-group: ~6–7

  • Aliphatic amine (Lys) R-group: ~10

  • Guanidinium (Arg) R-group: ~12

• For alanine, pI ≈ 6.0, calculated as $\text{pI} = \frac{pK<em>{a1} + pK</em>{a2}}{2} = \frac{2.3 + 9.7}{2} = 6.0$

Extra notes and context

• For isoelectric point calculations of other amino acids, use the rule: acidic amino acids have pI midway between their two COOH pKa values; basic amino acids have pI midway between the two amino group pKa values.

• Primary, secondary, tertiary, and quaternary structure concepts form the basis of understanding protein structure and function.

• This compilation mirrors the content from Dr. Jane Irwin’s course materials and linked references (e.g., LibreTexts, Khan Academy).