Zwitterion · pI · D/L · Essential AAs · Peptide bond · Protein structure · DNA/RNA
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Question 1
The α-amino acids found in proteins have:
A — both functional groups are on the same α-carbon. The α-carbon also bears a hydrogen and the variable R-group (side chain). This common backbone is what defines a standard protein amino acid. McMurry & Ballantine 8e Ch 28 §28.1; Stryer Biochemistry 8e §2.2
Question 2
At its isoelectric point (pI), an amino acid exists as:
B — Zwitterion. At pI the amino acid carries equal positive (+NH₃⁻) and negative (–COO⁻) charges, giving net charge = 0. It is not an uncharged molecule — both charges are present simultaneously. Net charge = 0 means no migration in an electric field. McMurry 8e §28.2; Stryer 8e §2.3
Question 3
The pI of an amino acid with pKa1 = 2.3 and pKa2 = 9.6 is:
C — pI = (pKa1 + pKa2) / 2 = 5.95. For a diprotic amino acid (simple, neutral R-group), pI is the average of the two pKas that flank the zwitterion species on a titration curve. For glycine: pKa1 = 2.3 (COOH), pKa2 = 9.6 (NH₃⁺); pI = 5.97. McMurry 8e §28.2; Lehninger 7e §3.3
Question 4
In Fischer projection, an L-amino acid has:
D — L-amino acid: –NH₂ on the LEFT. By analogy with L-glyceraldehyde (where –OH is on the left), L-amino acids have the amino group on the left in Fischer projection with –COOH at top. D-amino acids have –NH₂ on the right. Mnemonic: L = Left. McMurry 8e §28.2; Stryer 8e §2.2
Question 5
All naturally occurring protein amino acids are:
A — All L-configuration (except glycine which is achiral). This universal homochirality in proteins arose early in evolution and is essential for correct enzyme active-site geometry and protein folding. D-amino acids are found in some bacterial cell walls and certain peptide antibiotics. McMurry 8e §28.2; Lehninger 7e §3.2
Question 6
Essential amino acids CANNOT be:
B — Essential AAs cannot be synthesised by the body in adequate amounts; they must come from diet. They can still be used for protein synthesis (A), obtained from diet (C), and catabolised (D). There are 10 essential AAs: mnemonic PVT TIM HaLL — Phe, Val, Thr, Trp, Ile, Met, His, Arg, Leu, Lys. Lehninger 7e §3.2; Harper's Biochemistry 32e Ch 28
Question 7
PKU (phenylketonuria) is caused by deficiency of:
C — Phenylalanine hydroxylase (PAH). PAH normally converts phenylalanine → tyrosine. Without it, phenylalanine accumulates → alternative transamination to phenylpyruvate (a phenylketone) → urinary excretion and neurotoxicity. Neonatal screening (Guthrie test) detects raised Phe. Low-Phe diet prevents intellectual disability. Lehninger 7e §18.3; Harper's 32e Ch 29
Question 8
The mnemonic PVT TIM HaLL stands for:
D — The 10 essential amino acids:Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine, Lysine. (His and Arg are conditionally essential in infants.) These 10 must come from dietary protein. Lehninger 7e §3.2; Harper's 32e Ch 28
Question 9
Ninhydrin test detects:
A — Ninhydrin detects free α-amino groups, giving a characteristic purple (Ruhemann’s purple) colour. Proline (a secondary amine, cyclic pyrrolidine ring) gives a yellow colour instead. Used in amino acid analysis after paper/thin-layer chromatography. Reducing sugars → Benedict’s; disulfides → Ellman’s reagent. McMurry 8e §28.3; Stryer 8e §3.1
Question 10
The peptide bond is formed by:
B — Condensation reaction. The –COOH of one amino acid reacts with the –NH₂ of the next, releasing H₂O, to form the amide (–CO–NH–) peptide bond. Hydrolysis is the reverse (breaking) reaction, which requires acid, base, or protease enzymes. In cells, ribosomal peptidyl transferase catalyses the condensation. McMurry 8e §28.4; Stryer 8e §3.2
Question 11
The peptide bond has partial double-bond character because:
B — Resonance: N lone pair → C=O. The nitrogen lone pair is delocalized into the carbonyl, creating a C–N bond with ~40% double-bond character. Consequences: (1) the peptide bond is planar (6 atoms coplanar); (2) rotation about C–N is restricted; (3) two conformers exist (trans preferred, cis rare). This planarity is crucial for predicting protein secondary structure via Ramachandran plot (φ/ψ angles). McMurry 8e §28.4; Stryer 8e §3.2
Question 12
Protein primary structure refers to:
D — Primary structure = amino acid sequence. It is maintained by covalent peptide bonds (and disulfide bonds). 2° = local hydrogen-bonded patterns (helix, sheet). 3° = overall 3D fold. 4° = subunit assembly. Sanger determined insulin’s primary structure (1955, Nobel Prize). Anfinsen showed primary structure determines 3D folding. Stryer 8e §3.4; McMurry 8e §28.5
Question 13
The α-helix is stabilised by:
A — Backbone H-bonds, i to i+4. Each C=O of residue n hydrogen bonds to the N–H of residue n+4 (3.6 residues per turn). R-groups project outward. Proline disrupts α-helices (no N–H; rigid ring). β-sheets are stabilised by interstrand backbone H-bonds. Disulfide bonds stabilise tertiary structure, not secondary. Stryer 8e §3.5; McMurry 8e §28.5
Question 14
Sickle-cell anaemia results from:
B — Glu&sup6; → Val in β-globin. Glutamic acid (charged, hydrophilic) is replaced by valine (non-polar, hydrophobic). HbS molecules deoxygenated → expose a hydrophobic patch → polymerise into rigid fibres → distort RBCs into sickle shape → haemolysis and vascular occlusion. Classic example of how a single amino acid change (primary structure) causes catastrophic functional disease. Stryer 8e §7.2; Lehninger 7e §5.5
Question 15
Purines (double-ring nitrogenous bases) are:
C — Adenine (A) and Guanine (G) are purines. Purines have a fused bicyclic structure (imidazole ring fused to pyrimidine ring). Pyrimidines are single-ring: Cytosine (B), Thymine (T, DNA only), Uracil (U, RNA only). Memory aid: PURe As Gold (PURines = Adenine, Guanine). McMurry 8e §28.6; Stryer 8e §1.2
Question 16
Pyrimidines (single-ring) present in DNA are:
B — DNA pyrimidines: Cytosine and Thymine. RNA replaces thymine with uracil (C and U are the RNA pyrimidines). Thymine is 5-methyluracil; the methyl group adds stability to DNA (fewer deamination errors). All four bases in DNA: A, G (purines) + C, T (pyrimidines). RNA: A, G + C, U. McMurry 8e §28.6; Stryer 8e §4.1
Question 17
Adenine pairs with Thymine via:
B — A=T: 2 hydrogen bonds; G≡C: 3 hydrogen bonds. Watson-Crick base pairing is antiparallel and complementary. G≡C has one extra H-bond (amino group on C3 of G ↔ carbonyl on C2 of C), making it stronger. This is why GC-rich DNA is harder to denature (higher Tm). McMurry 8e §28.7; Stryer 8e §4.2
Question 18
A DNA strand with higher G+C content has:
B — Higher G+C → higher Tm. G≡C pairs have 3 H-bonds vs A=T’s 2; more energy required to separate the strands (denature/melt). Tm also increases with strand length. This is exploited in PCR primer design: GC-rich primers bind more tightly. Human genomic DNA (~40% GC) has Tm ~87°C in standard salt. Stryer 8e §4.2; Lehninger 7e §24.1
Question 19
RNA differs from DNA in:
C — Three key differences: uracil/thymine, ribose/deoxyribose, ss/ds. RNA sugar is ribose (2′-OH present); DNA has deoxyribose (2′-H). The 2′-OH makes RNA less stable (susceptible to alkaline hydrolysis). RNA is usually single-stranded but can fold with intramolecular base pairing (tRNA, rRNA). mRNA, tRNA, rRNA, snRNA are all RNA types. McMurry 8e §28.7; Stryer 8e §4.5
Question 20
Which levels of protein structure are disrupted first by urea or heat?
D — Denaturation disrupts 2°, 3° (and 4°) structure first. Primary structure consists of covalent peptide bonds, which require strong chemical hydrolysis to break (not urea or heat). Urea breaks H-bonds and hydrophobic interactions; heat increases kinetic energy, disrupting weak non-covalent forces. Disulfide bonds (–S–S–) require reducing agents (e.g. β-mercaptoethanol) to cleave. Denaturation is usually irreversible in vivo (exception: Anfinsen’s ribonuclease refolding in vitro). Stryer 8e §3.6; McMurry 8e §28.5
1. Zwitterion▼
The dipolar ionic form of an amino acid at its isoelectric point (pI). Carries a protonated amino group (+NH₃⁻) and a deprotonated carboxylate (–COO⁻) simultaneously; net charge = 0. Predominant form in physiological conditions (pH ~7.4). Does not migrate in an electric field. Not the same as a neutral uncharged molecule — both charges are present. McMurry 8e §28.2; Stryer 8e §2.3
2. Isoelectric point (pI)▼
The pH at which an amino acid (or protein) has zero net charge and exists predominantly as the zwitterion. For simple (neutral R-group) amino acids: pI = (pKa1 + pKa2) / 2 (average of the two pKas flanking the zwitterion). Determines electrophoretic mobility — at pI, the molecule does not move in an electric field. Used in isoelectric focusing (IEF) to separate proteins. Acidic amino acids (Asp, Glu) have low pI; basic ones (Lys, Arg) have high pI. McMurry 8e §28.2; Lehninger 7e §3.3
3. Essential amino acid▼
An amino acid that cannot be synthesised by the human body in adequate amounts and must be obtained from dietary protein. There are 10: PVT TIM HaLL (Phe, Val, Thr, Trp, Ile, Met, His, Arg, Leu, Lys). Histidine and Arginine are conditionally essential in infants. Deficiency causes growth retardation and negative nitrogen balance. Non-essential AAs (e.g. Ala, Gly, Ser) can be synthesised from metabolic intermediates. Lehninger 7e §3.2; Harper’s 32e Ch 28
4. Peptide bond▼
An amide bond (–CO–NH–) formed by condensation between the α-carboxyl group of one amino acid and the α-amino group of the next, with release of water. Has partial double-bond character due to resonance of the N lone pair into C=O → the peptide unit is planar and rigid (rotation restricted). Trans configuration strongly preferred. Hydrolysed by acid, alkali, or proteases. McMurry 8e §28.4; Stryer 8e §3.2
5. Denaturation▼
The disruption of a protein’s secondary, tertiary, and quaternary structure by agents such as heat, extremes of pH, urea, guanidinium chloride, detergents, or organic solvents. Non-covalent interactions (H-bonds, hydrophobic interactions, electrostatic bonds) are broken; primary structure (covalent peptide bonds) remains intact. Usually irreversible in vivo. The protein loses its native conformation and biological activity. Anfinsen’s experiment (ribonuclease) showed that primary structure encodes all folding information. Stryer 8e §3.6; McMurry 8e §28.5
6. Purine▼
A double-ring nitrogenous base consisting of a pyrimidine ring fused to an imidazole ring. The two purines in DNA/RNA are Adenine (A) and Guanine (G). A pairs with T (in DNA) or U (in RNA) via 2 hydrogen bonds; G pairs with C via 3 hydrogen bonds. Higher G+C content → higher DNA melting temperature (Tm). Memory: PURe As Gold. Pyrimidines (single ring): C, T (DNA), U (RNA). McMurry 8e §28.6; Stryer 8e §1.2
E1. Define isoelectric point (pI) and explain how to calculate it for glycine (pKa1=2.3, pKa2=9.6). Draw the ionic species at pH 1, pH 6, and pH 12. (7 marks)Essay
Definition: pI is the pH at which an amino acid carries zero net charge and exists as the zwitterion (+NH₃–CHR–COO⁻). It does not migrate in an electric field.
Calculation for glycine: pI = (pKa1 + pKa2) / 2 = (2.3 + 9.6) / 2 = 5.95. This is the average of the two pKas flanking the zwitterion form on the titration curve.
Clinical relevance: Isoelectric focusing separates proteins by pI in a pH gradient. Haemoglobin variants (HbA vs HbS) differ in pI due to Glu→Val substitution and can be separated electrophoretically.
Cite: McMurry 8e §28.2; Lehninger 7e §3.3; Stryer 8e §2.3
E2. Explain the L and D configuration of amino acids. Why are all protein amino acids L? Describe the clinical significance of D-amino acids in bacteria. (6 marks)Essay
L/D configuration: Based on Fischer projection with –COOH at top and –R at bottom. If –NH₂ is on the left → L; on the right → D. By analogy with L-glyceraldehyde (–OH on left) and D-glyceraldehyde (–OH on right).
Absolute configuration: Most L-amino acids are (S) in R/S nomenclature (except cysteine, which is R due to sulfur's high atomic mass changing priority order).
Why all L in proteins?
Evolved through symmetry breaking in prebiotic chemistry (random initial enrichment, amplified by autocatalysis).
Ribosomal peptidyl transferase active site is stereospecific for L-amino acyl-tRNAs.
A D-amino acid cannot be correctly accommodated in the ribosomal A-site.
Homochirality is essential for regular protein secondary structures (all backbone torsion angles in L-amino acid range on Ramachandran plot).
D-amino acids in bacteria (clinical significance):
Bacterial cell walls (peptidoglycan) contain D-glutamate and D-alanine.
Human proteases cannot hydrolyse D-peptide bonds → the cell wall resists host defence enzymes.
Vancomycin targets the D-Ala–D-Ala terminus of peptidoglycan precursors; vancomycin-resistant enterococci (VRE) substitute D-Lac for D-Ala.
Cite: McMurry 8e §28.2; Stryer 8e §2.2; Murray RK Harper’s 32e Ch 28
E3. Describe the four levels of protein structure. Give one stabilising force at each level and one clinical example of a protein whose structure-function is well understood. (8 marks)Essay
1° Primary structure — the linear sequence of amino acids joined by peptide bonds.
Stabilised by: covalent peptide bonds (+ disulfide bonds between cysteines).
Example: Insulin (51 AAs in 2 chains); Sanger’s insulin sequencing (1955) first proved sequence determines function.
2° Secondary structure — local hydrogen-bonded patterns of the backbone: α-helix (H-bond: C=On to N–Hn+4) and β-sheet (parallel or antiparallel interstrand H-bonds).
Stabilised by: backbone H-bonds.
Example: Keratin (hair, nails) is a coiled-coil α-helical fibrous protein; mutations in keratin cause epidermolysis bullosa.
3° Tertiary structure — overall 3D fold of a single polypeptide chain; brings distant segments into proximity.
Stabilised by: hydrophobic interactions (non-polar R-groups buried in core), H-bonds, electrostatic (ionic) interactions, disulfide bridges, van der Waals forces.
Example: Myoglobin (153 AAs, 8 α-helices, 1 haem group); first protein structure solved by Kendrew & Perutz by X-ray crystallography (1958).
4° Quaternary structure — assembly of two or more polypeptide subunits (protomers).
Stabilised by: same forces as 3° (hydrophobic interactions, H-bonds, electrostatic interactions at subunit interfaces).
Example: Haemoglobin (2α2β tetramer); cooperative O₂ binding and allosteric regulation (Bohr effect, BPG); sickle-cell Hb polymerises via hydrophobic patch exposed by Glu&sup6;→Val mutation.
Cite: Stryer 8e §3.4–3.7; McMurry 8e §28.5; Lehninger 7e §4.1–4.4
E4. Explain the molecular basis of sickle-cell anaemia. What single amino acid change is involved, and how does it alter the physicochemical properties of haemoglobin? (6 marks)Essay
Mutation: A single point mutation in the β-globin gene: codon 6 changes from GAG (Glu) to GTG (Val). This substitutes a charged, hydrophilic glutamic acid (acidic, –CH₂CH₂COO⁻) with a small, non-polar valine (–CH(CH₃)₂).
Physicochemical change:
Glu⁼ at position 6 is on the surface of β-globin; its negative charge is important for keeping HbA soluble.
Val substitution creates a hydrophobic “sticky patch” on the exterior of the β chain.
HbS moves faster toward the anode than HbA on electrophoresis (lower pI: loss of negative charge).
Polymerisation → sickling:
In the deoxygenated state, HbS undergoes a conformational shift that exposes the Val⁼ hydrophobic patch.
This patch fits into a complementary hydrophobic pocket on an adjacent HbS molecule → nucleation and polymerisation into long insoluble fibres (HbS tactoids).
Fibres distort RBCs into rigid sickle shapes → haemolysis (anaemia) and vascular occlusion (painful crises, organ damage).
Cite: Stryer 8e §7.2; Lehninger 7e §5.5; Harper’s 32e Ch 7
E5. Compare the structures of DNA and RNA. Explain Watson-Crick base pairing, state the number of hydrogen bonds for each pair, and explain why GC-rich DNA has a higher melting temperature. (7 marks)Essay
Structural comparison:
Sugar: DNA = 2′-deoxyribose (no 2′-OH); RNA = ribose (2′-OH). The 2′-OH makes RNA susceptible to alkaline hydrolysis and limits RNA to a single strand in most contexts.
Bases: DNA = A, G, C, T (thymine); RNA = A, G, C, U (uracil). Thymine is 5-methyluracil — the methyl group stabilises DNA and reduces spontaneous deamination errors.
Strandedness: DNA = double-stranded antiparallel helix (B-form: 10 bp/turn, 3.4 nm pitch, 2 nm diameter). RNA = usually single-stranded but can fold (tRNA cloverleaf, rRNA hairpins).
A = T (A = U in RNA): 2 hydrogen bonds. Adenine N6–H···O4 of thymine + N1···H–N3 of thymine.
G ≡ C: 3 hydrogen bonds. Guanine O6···H–N4(C) + N1–H···N3(C) + N2–H···O2(C). The extra H-bond makes G≡C significantly stronger than A=T.
Base pairs are complementary (A only with T/U; G only with C) and antiparallel (5′→3′ on one strand, 3′→5′ on the other).
Why GC-rich DNA has a higher Tm (melting temperature):
Tm = temperature at which 50% of DNA double strands are separated (denatured).
G≡C has 3 H-bonds vs A=T’s 2 H-bonds → each G≡C pair requires more energy to disrupt.
DNA with 50% GC has Tm ~87°C; 100% GC (theoretical) would have even higher Tm; 100% AT would have lower Tm.
Rule of thumb: each 1% increase in GC content raises Tm by ~0.4°C.
Application: PCR primer design — GC-rich primers have higher melting temperatures; primers must have similar Tm to anneal at the same temperature. Also relevant to understanding gene expression in thermophilic organisms.
Cite: McMurry 8e §28.6–28.7; Stryer 8e §4.1–4.2; Lehninger 7e §24.1