Biochemistry Essentials: Protein Folding, Hemoglobin, and Glycan Structure

Posted on Dec 6, 2025 in Biochemistry

Protein Structure and Function Fundamentals

Proteins are at the center of all biological processes. They catalyze reactions, regulate pathways, transport molecules, and form most of the structural framework of cells.

Core Principle: Structure → Function.

Historical Milestones in Protein Science

• Hsien Wu (1931): Showed that denaturation destroys non-covalent interactions, leading to unfolding and loss of function.
• Bernal & Hodgkin (1934): X-ray diffraction of pepsin revealed that proteins are ordered, crystalline molecules.
• Kendrew (1958): Produced the first atomic model (sperm-whale myoglobin), revealing that proteins have specific but irregular three-dimensional folds.

Four Levels of Protein Structure

🧠 Mini-Summary: Proteins aren’t random blobs — they’re precisely folded machines. Understanding their four structural levels explains nearly every biological function.

4.1 Secondary Protein Structure

4.1A The Peptide Bond and Torsion Angles

• The peptide bond has approximately 40% double-bond character, making it planar and rigid.
• Cis vs. Trans: The *trans* configuration is favored (~8 kJ/mol more stable); *cis* mainly occurs before **Proline**.
• Rotation occurs only about φ (phi) = Cα–N and ψ (psi) = Cα–C.
• Certain φ/ψ combinations cause steric clashes, which are visualized on a Ramachandran plot.
  • Allowed regions include the α-helix, β-sheet, and polyproline (PII) helix.
  • Glycine: Very flexible (large allowed area).
  • Proline: Rigid (limited φ ≈ –60°).

🧠 Mini-Summary: Peptide bonds are flat, and protein folding is basically a dance of φ and ψ angles that avoid steric crashes.

4.1B Regular Secondary Structures (Alpha Helices & Beta Sheets)

The Alpha (α) Helix

• Discovered by Pauling & Corey (1951).
• Right-handed; 3.6 residues/turn, pitch = 5.4 Å.
• H-bond: C=O of residue n → N–H of n + 4.
• Side chains project out and back; hydrophobic/hydrophilic patterns create amphipathic helices.
• Common residues: Alanine, Leucine, Methionine. Breakers: Proline and Glycine.

Beta (β) Sheets

• Chains (β-strands) are H-bonded side-by-side.
  • Antiparallel: Strong, linear H-bonds.
  • Parallel: Weaker, angled H-bonds.
• Pleated appearance (~7 Å repeat); side chains alternate up/down.
• Found in silk fibroin and immunoglobulins.

Turns, Loops, and the Polyproline II Helix

• β-turns: Reverse direction; typically 4 residues; often contain Proline (residue 2) and Glycine (residue 3).
• Ω-loops: Flexible surface connectors.
• Polyproline II (PII) helix: Left-handed (~3 residues/turn), dominant in collagen.

🧠 Mini-Summary: α-Helices coil inward, β-sheets stretch outward, and turns connect them. Together, they form the protein’s “scaffolding.”

4.1C Fibrous vs. Globular Proteins

Alpha-Keratin Structure and Coiled Coils

• Two right-handed α-helices form a left-handed coiled-coil.
• A 7-residue pseudorepeat (a-b-c-d-e-f-g) where residues ‘a’ and ‘d’ are hydrophobic forms a “stripe” that zips the coils together.
• Cross-linked by disulfide bonds (Cysteine–Cysteine); hardness depends on the number of cross-links.
• Hierarchy: dimer → protofilament → protofibril → microfibril → macro-fiber.

Collagen: The Triple Helix

• Triple helix: Three left-handed PII-like chains form a right-handed superhelix.
• Repeating sequence: Gly–X–Y (X = Proline, Y = Hydroxyproline (Hyp)).
• Glycine fits in the core; H-bonds form between chains (Gly N–H → Pro C=O).
• Hydroxylation of Proline and Lysine requires Vitamin C; deficiency causes scurvy.
• Cross-linked by allysine (formed via lysyl oxidase).

🧠 Mini-Summary: Keratin provides coiled helix strength; Collagen provides triple helix toughness. Both use H-bonds and cross-links to resist stress.

4.2 Tertiary Protein Structure

4.2A Structural Motifs and Domains

• Motifs (supersecondary structures): Recurring combinations of helices and sheets.
  • βαβ motif – common in enzymes.
  • β-hairpin – antiparallel link.
  • αα motif – packed helices.
  • Greek key – folded β-hairpin forming a 4-strand sheet.
• Domains: Independent folding units (~40–200 amino acids) with specific functions.
  • e.g., Rossmann fold for NAD⁺ binding in dehydrogenases.

4.2B Side-Chain Distribution and Hydrophobic Core

• Non-polar residues (Valine, Leucine, Isoleucine, Methionine, Phenylalanine) cluster in the protein interior.
• Charged residues (Arginine, Lysine, Aspartate, Glutamate) are typically found on the surface.
• Uncharged polar residues (Serine, Threonine, Asparagine, Glutamine) are on the surface or H-bonded inside.

4.2C Forces Stabilizing Tertiary Structure

1. Hydrophobic effect (the primary entropic driver).
2. Hydrogen bonds (fine-tuning specificity).
3. Ionic interactions (salt bridges, mostly on the surface).
4. Metal ions (e.g., Zn²⁺ in zinc fingers).
5. Disulfide bonds (Cysteine–Cysteine covalent links).
6. Molecular crowding favors compact folds in cells.

🧠 Mini-Summary: Tertiary structure is defined by a hydrophobic core held together by a web of weak forces and occasional metal or disulfide “glue.”

4.3 Quaternary Structure and Protein Complexes

4.3A Subunits, Protomer, and Interfaces

• Oligomer: A protein composed of multiple subunits.
• Protomer: The repeating functional unit.
• Subunits interact via hydrophobic and van der Waals forces, often supplemented by salt bridges.
• Advantages of multisubunit design: easier assembly, defect replacement, and regulation.

4.3B Symmetry in Oligomeric Proteins

• Cyclic symmetry (C_n): One rotational axis (C₂, C₃ …).
• Dihedral symmetry (D_n): An n-fold axis perpendicular to a 2-fold axis (e.g., D₂ is most common).
• Examples:
  • Hemoglobin = α₂β₂ heterotetramer.
  • Virus capsids = icosahedral (D₃, D₅ etc.).
  • Keratin fibers = parallel coiled coils.

4.3C Large Functional Protein Assemblies

• Enzyme assemblies (e.g., pyruvate dehydrogenase complex).
• Molecular machines: ATP synthase (F₁F₀) is a multi-subunit rotary engine.

🧠 Mini-Summary: Quaternary structure lets proteins work as teams — each subunit amplifies or regulates the others.

4.4 Protein Folding, Denaturation, and Chaperones

4.4A Protein Stability and Denaturation

• Proteins are marginally stable (~0.4 kJ/mol per amino acid).
• Denaturants: Heat, pH shifts, detergents, urea/guanidinium.
• Anfinsen (1957): Showed that RNase A renatures, proving that the amino acid sequence determines the final structure.

4.4B Folding Pathways and the Folding Funnel

1. Secondary structure forms (5 ms).
2. Molten globule (5–1000 ms): Compact hydrophobic core forms.
3. Tertiary rearrangement (seconds).

• Folding funnel: ΔH decreases, ΔS decreases, leading toward the stable native state.

4.4C Molecular Chaperones and Assisted Folding

• PDI (Protein Disulfide Isomerase): Rearranges incorrect disulfide bonds.
• Molecular Chaperones:
  • Hsp70 + Hsp40: Prevent aggregation of nascent chains.
  • GroEL/ES (Chaperonin): An ATP-driven “cage” for refolding proteins.
    • Requires 7 ATP per cycle; folding occurs in the *cis* ring while the *trans* ring resets.

🧠 Mini-Summary: Folding is a guided process with helpers — like a factory assembly line that uses energy (ATP) to produce correctly folded products.

4.5 Myoglobin and Hemoglobin: Oxygen Transport

(Biochemistry in Focus)

Myoglobin (Mb): Oxygen Storage

• Monomeric, 153 amino acids, mostly α-helical.
• Contains a heme (Fe²⁺) group for O₂ binding.
• Histidine residues coordinate iron and O₂.
• Functions:
  • Facilitates O₂ diffusion in muscle.
  • Stores O₂ in diving mammals.
• Binding curve: Hyperbolic ( Y = pO₂ / (p₅₀ + pO₂) ) where p₅₀ ≈ 2.8 torr.

🧠 Mini-Summary: Myoglobin is the simple O₂ bucket — one heme, one O₂, no cooperativity.

Hemoglobin (Hb): Cooperative Oxygen Transport

• Tetramer: α₂β₂ (a heterotetramer, or dimer of αβ protomers).
• Two states:
  • T (Tense): Low affinity, deoxy form.
  • R (Relaxed): High affinity, oxy form.
• Cooperative binding: Sigmoidal curve (Hill coefficient ≈ 3).
  • O₂ binding to one heme shifts Fe²⁺ into the plane, triggering the T → R transition.
• Bohr Effect: Decreased pH or increased CO₂ favors the T state, promoting O₂ release.
• BPG (2,3-bisphosphoglycerate): Binds in the central cavity of the T state, lowering O₂ affinity, which is essential for efficient O₂ delivery.
• Sickle-Cell Anemia: β6 Glutamate → Valine mutation causes hydrophobic aggregation of deoxy HbS, leading to fiber formation and red blood cell sickling.
  • Heterozygotes resist malaria (*Plasmodium falciparum*).

🧠 Mini-Summary: Hemoglobin is a team player — subunits cooperate for O₂ transport and regulation. Mutations in its structure illustrate how “structure = function.”

Key Takeaways: Protein Structure and Function

Chapter 5: Carbohydrates and Glycobiology

5.1A Monosaccharides: Aldoses and Ketoses

🧠 Mini-Summary: Sugars are polyhydroxy aldehydes (aldoses) or ketones (ketoses) with three or more carbons. They are named by carbonyl type plus carbon count (triose…hexose). D/L configuration is set by the chiral center farthest from the carbonyl (matching D/L-glyceraldehyde). Epimers differ at one stereocenter (Glc/Man @ C2; Glc/Gal @ C4). Ketohexoses have one fewer chiral center than aldohexoses.

• Definition: Monosaccharides are aldehyde/ketone derivatives of straight-chain polyhydroxy alcohols with three or more carbons.
  • Aldose (aldehyde at C1) vs. ketose (ketone, most commonly at C2).
  • Trioses, tetroses, pentoses, hexoses, heptoses… by carbon count.
• Stereochemistry:
  • D/L assigned by configuration at the chiral center farthest from the carbonyl (match to D/L-glyceraldehyde; Fischer projection).
  • D-glucose (an aldohexose) has 4 chiral centers (C2–C5), leading to 2⁴ = 16 stereoisomers.
  • Epimers: Differ at one chiral center (e.g., D-glucose vs. D-mannose at C2; D-glucose vs. D-galactose at C4).
• Common aldoses: D-glucose, D-mannose, D-galactose; ribose (RNA) and glyceraldehyde (metabolism).
• Common ketoses: Dihydroxyacetone, ribulose, fructose. A ketohexose has 2³ = 8 stereoisomers (4 D, 4 L).

5.1B Configuration, Conformation, and Cyclization

🧠 Mini-Summary: Carbonyl + alcohol forms a cyclic hemiacetal/hemiketal (pyranose or furanose). Cyclization creates an anomeric carbon: α (anomeric OH opposite CH₂OH) vs. β (same side). Mutarotation interconverts these forms via the open chain (D-Glucose equilibrium ≈ β 64% / α 36%). Rings are not planar—chair forms dominate; β-D-glucopyranose is extra stable because all bulky groups are equatorial.

• Cyclization: Intramolecular formation of hemiacetal (aldose) or hemiketal (ketose) results in cyclic sugars.
  • Pyranose (6-membered; e.g., glucopyranose) and furanose (5-membered; e.g., fructofuranose).
• Anomers: A new chiral center is created at the anomeric carbon.
  • α = Anomeric OH opposite the CH₂OH (at the stereocenter defining D/L; C5 in hexoses).
  • β = Anomeric OH on the same side as CH₂OH.
  • Mutarotation: α ⇌ open chain ⇌ β in water; D-glucose at equilibrium ≈ β 63.6% / α 36.4%; linear form is trace.
• Conformation: Rings are not planar (due to sp³ centers).
  • Pyranoses adopt chair conformers; bulky groups prefer equatorial. β-D-glucose places all five non-H substituents equatorial, contributing to its stability and natural abundance.
  • Furanoses also pucker; interconversion of conformations is fast, but configurational changes (α↔β, furanose↔pyranose, epimers) require bond breaking (enzyme-catalyzed in cells).

5.1C Sugar Modifications and Glycosidic Bonds

🧠 Mini-Summary: Aldoses oxidize to aldonic acids (C1) or uronic acids (C6); reduction yields alditols. Common variants include deoxy (2-deoxyribose) and amino sugars (GlcN, GalN; sialic acids). The anomeric carbon forms glycosidic bonds (O/N); once bonded, mutarotation is prevented. Reducing sugars have a free anomeric carbon. Polysaccharides can be linear or branched; linkage identity (α/β, C→C) dictates structure and function.

• Oxidation:
  • Aldehyde → aldonic acid (e.g., gluconic acid).
  • Primary alcohol (C6) → uronic acid (e.g., D-glucuronic acid).
• Reduction: Carbonyl → alditol (e.g., ribitol (flavins), glycerol, myo-inositol (lipids), xylitol sweetener).
• Deoxy sugars: 2-deoxyribose (DNA); L-fucose is a common L-sugar in glycans.
• Amino sugars: Glucosamine, galactosamine; N-acetylneuraminic acid (Neu5Ac; sialic acid) is important in glycoproteins/glycolipids.
• Glycosidic bonds: Anomeric C + alcohol (O-glycoside) or amine (N-glycoside) forms α/β linkages.
  • These bonds are stable to hydrolysis without enzymes; the anomeric C is locked (no mutarotation).
  • Sugars with a free anomeric carbon are reducing sugars (they can reduce Cu²⁺ → Cu⁺).
• Polysaccharides (glycans): Can be homo- vs. hetero-; they can branch (due to multiple OH linkage positions).
  • Full description requires identifying the components, anomeric forms, and linkages; characterized using specific exo/endo-glycosidases and NMR.

5.2 Disaccharides and Polysaccharides

5.2A Common Disaccharides: Lactose and Sucrose

🧠 Mini-Summary: Lactose = β-Galp(1→4)Glc (reducing). Sucrose = α-Glcp(1→2)β-Fruf (nonreducing, as both anomeric carbons are involved in the bond). Reading the name tells you the anomer, carbons linked, and ring type.

• Lactose: β-D-Galp-(1→4)-D-Glcp; it is reducing (due to the free anomeric carbon on glucose).
• Sucrose: α-D-Glcp-(1→2)-β-D-Fruf; it is nonreducing (both anomeric carbons are involved in the bond).
• Oligosaccharides (3–10 residues) are rarer (mostly found in plants).

5.2B Storage Polysaccharides: Starch and Glycogen

🧠 Mini-Summary: Storage uses α-linkages (enzyme-friendly). α-Amylose = linear α(1→4) helix; amylopectin = α(1→4) with α(1→6) branches every 24–30 residues. Glycogen is like amylopectin but more branched (every 8–14 residues), enabling rapid mobilization from many nonreducing ends by glycogen phosphorylase.

• Starch (plants): A mixture of:
  • α-amylose: Linear α(1→4); helical; thousands of residues.
  • Amylopectin: α(1→4) chains with α(1→6) branches every 24–30 residues; up to 10⁶ glucose units.
  • Digestion: Salivary & pancreatic amylase (random α(1→4) hydrolysis) yields oligosaccharides; α-glucosidase trims; debranching enzyme handles α(1→6) linkages.
  • There is one reducing end per polymer (starch overall is reducing but functionally has many nonreducing ends).
• Glycogen (animals): Like amylopectin but more branched—α(1→6) every 8–14 residues.
  • Mobilization: Glycogen phosphorylase removes α(1→4) units from nonreducing ends; debranching enzyme resolves branches, leading to rapid glucose release.

5.2C Structural Polysaccharides: Cellulose and Chitin

🧠 Mini-Summary: Structure uses β-linkages, resulting in extended, H-bonded sheets. Cellulose = β(1→4) Glc, forming flat ribbons that create strong, insoluble fibers (no vertebrate cellulase; microbes digest it). Chitin = β(1→4) N-acetyl-D-glucosamine (GlcNAc), with cellulose-like packing; major component of exoskeletons and fungal walls.

• Cellulose: Linear β(1→4) D-glucose (up to ~15,000 residues). Each ring is rotated 180°, forming flat ribbons; extensive intra/inter-chain H-bonding creates sheets → stacks; insoluble, strong; embedded in a matrix with hemicellulose & lignin in plant walls.
  • No vertebrate enzyme for β(1→4) glucan hydrolysis; microbial cellulases (in herbivores/termites) digest it slowly.
  • Contrast: α(1→4) linkages in starch lead to helical/irregular structures, while β(1→4) linkages in cellulose lead to extended/packed structures.
• Chitin: β(1→4) N-acetyl-D-glucosamine; major component in arthropod exoskeletons & fungal walls; similar packing to cellulose.

5.2D Glycosaminoglycans (GAGs) and Hydrated Gels

🧠 Mini-Summary: Extracellular matrix (ECM) gels composed of alternating uronic acid + hexosamine units; highly anionic, binding water and cations to create viscoelastic properties. Hyaluronate (n is huge, nonsulfated) acts as a shock absorber/lubricant. Sulfated GAGs (chondroitins, dermatan, keratan, heparin/heparan) tune signaling and anticoagulation. Plants use pectin (Ca²⁺-cross-linked gels). Biofilms are hydrated bacterial polysaccharide matrices that protect cells.

• Hyaluronate: (GlcA-β(1→3)-GlcNAc)_n with β(1→4) between repeats; n ≈ 80–8000; viscoelastic and exhibits shear-dependent viscosity, used for joint lubrication and vitreous humor.
• Sulfated GAGs: Disaccharide repeats (50–1000 units):
  • Chondroitin-4/6-sulfate (GalNAc sulfation differs).
  • Dermatan sulfate (C5 epimerization of GlcA → IdoA).
  • Keratan sulfate (heterogeneous; variable sulfate; includes fucose, mannose, GlcNAc, sialic acid).
  • Heparin: Most highly sulfated (~2.5 sulfates/disaccharide); a potent anticoagulant; stored in mast cell granules.
  • Heparan sulfate: Related but less sulfated/more N-acetyl; found on the cell surface & ECM; specific sulfation codes regulate growth factor signaling (development, wound healing).
• Plant analog: Pectin (α(1→4) galacturonate with rhamnose + side chains; Ca²⁺ cross-linking) forms gels (jams/jellies).
• Bacterial biofilms: Hydrated polysaccharide matrices (e.g., poly-GlcA, PNAG, cellulose-like) provide adhesion, desiccation, and antibiotic protection; problematic on medical devices.

5.3 Glycoconjugates: Structure and Recognition

5.3A Proteoglycans and Cartilage Resilience

🧠 Mini-Summary: A Hyaluronate backbone binds many core proteins bearing long GAG chains (keratan/chondroitin) to form gigantic bottlebrush complexes. Highly hydrated and polyanionic, they provide cartilage resilience (pressure expels water until charge repulsion halts compression; water returns on release).

• Architecture: Hyaluronate backbone + link protein + many core proteins.
  • Each core protein carries dozens of keratan sulfate and chondroitin sulfate chains (attached via O-linkages to Serine/Threonine through short oligosaccharides).
  • Also contains N-linked oligosaccharides near the attachment site.
• Scale: The central HA (0.4–4 μm) can bind up to ~100 core proteins; each with ~50 KS (≤250 disaccharides) and ~100 CS (≤1000 disaccharides), forming mega-Dalton complexes.
• Function: Highly hydrated gels; responsible for cartilage resilience. Movement nourishes avascular cartilage.

5.3B Peptidoglycan and Bacterial Cell Walls

🧠 Mini-Summary: GlcNAc–MurNAc β(1→4) chains are cross-linked by tetrapeptides (containing D-amino acids) to form a mesh that prevents osmotic lysis. This structure is targeted by lysozyme and many antibiotics. Gram-positive bacteria have a thick peptidoglycan layer; Gram-negative bacteria have a thin layer plus an outer membrane.

• Backbone: Repeating β(1→4) GlcNAc–MurNAc; the MurNAc lactyl group is amide-linked to a tetrapeptide (contains D- and L-amino acids; species-dependent).
• Cross-linking: Peptide bridges between chains (e.g., *S. aureus*: pentaglycine bridge from D-Alanine to Lysine ε-NH₃⁺).
• Defense/Therapy: D-amino acids resist common proteases; lysozyme hydrolyzes the β(1→4) GlcNAc–MurNAc bond; multiple antibiotics block biosynthesis.
• Gram Status:
  • Gram-positive: Thick peptidoglycan (~25 nm) → retains crystal violet stain.
  • Gram-negative: Thin peptidoglycan (~3 nm) + outer membrane → does not retain crystal violet.

5.3C Glycosylation of Eukaryotic Proteins (N- and O-linked)

🧠 Mini-Summary: N-linked (Asn-X-Ser/Thr): Transfer of Glc₃Man₉GlcNAc₂ occurs in the ER, followed by trimming and rebuilding in the Golgi. All share the Man₃GlcNAc₂ core; outcomes vary (microheterogeneity). O-linked (Ser/Thr): Starts with GalNAc-O-Ser/Thr in the Golgi; sites are set by local structure; chains can be short (e.g., collagen) or very long (proteoglycans).

• N-linked (Asn-X-Ser/Thr; X≠Pro, rarely Asp/Glu/Leu/Trp):
  1. Transfer of a preassembled oligosaccharide (Glc₃Man₉GlcNAc₂) to Asparagine during translation (ER lumen).
  2. Trimming by glucosidases/mannosidases (ER→Golgi).
  3. Rebuilding by glycosyltransferases (adding GlcNAc, Gal, Fuc, sialic acid, etc.) results in high-mannose or complex types.
  • All share a common core pentasaccharide (Man₃GlcNAc₂).
  • Results vary by protein/cell, leading to microheterogeneity.
• O-linked (Ser/Thr OH):
  • Initiated by α-GalNAc transfer → typical core β-Gal-(1→3)-α-GalNAc-O-Ser/Thr; elongated in the Golgi; no strict sequence motif (site dictated by local structure).
  • Ranges from single sugars (e.g., collagen) to long chains (e.g., proteoglycans).

5.3D Oligosaccharide Roles in Recognition and Function

🧠 Mini-Summary: Carbohydrate chains shield, stabilize, and shape proteins; they also encode recognition motifs read by lectins (e.g., selectins in leukocyte rolling). ABO antigens are terminal oligosaccharide variants on glycoproteins/lipids (H → +GalNAc = A; +Gal = B; inactive transferase = O); mismatched transfusions cause agglutination.

• Structural roles: Surface shielding, resistance to proteolysis; restriction of backbone flexibility; aid in folding (N-linked, cotranslational); O-linked clusters can stiffen/extend segments.
• Recognition: Cell surfaces are sugar-coated (the glycocalyx).
  • Lectins bind specific sugar motifs via H-bond networks plus CH/π stacking with aromatics.
  • Selectins mediate leukocyte–endothelium adhesion during inflammation by recognizing defined oligosaccharides.
  • Gamete recognition and pathogen entry often rely on carbohydrate–protein binding.
• Clinical—ABO antigens: Nonreducing-end oligosaccharide variants on glycoproteins/glycolipids.
  • H antigen precursor → A adds GalNAc (A-transferase), B adds Gal (B-transferase; differs by 4 amino acids from the A enzyme), O enzyme is inactive (truncated).
  • Mismatch leads to antibody-mediated agglutination.

Quick Definitions (Exam Favorites)

• Reducing sugar: Has a free anomeric carbon; can reduce Cu²⁺.
• Anomeric carbon: The carbonyl carbon that becomes chiral upon ring closure (C1 in aldoses, C2 in ketoses like fructose).
• Epimer: Stereoisomers differing at one chiral center.
• Mutarotation: α↔β interconversion via the open chain in water.
• Glycosidic linkage notation: Donor anomer/monosaccharide-ring, (C#→C#), acceptor (e.g., β-Galp-(1→4)-Glcp).

High-Yield Comparisons

• Starch/Glycogen (α) = Energy storage; helical, enzyme-accessible.
• Cellulose/Chitin (β) = Structure; extended sheets, insoluble, strong.
• Proteoglycan vs. Glycoprotein:
  • Proteoglycan = Protein + long GAGs (highly anionic; ECM gel; bottlebrush).
  • Glycoprotein = Protein + short N/O-linked oligosaccharides (diverse functions; recognition).

Self-Check Problem Set

1. Is lactose reducing or nonreducing? Reducing (free anomeric C on glucose).
2. Which anomer of D-glucose places all substituents equatorial? β-D-glucopyranose.
3. Why is heparin such a strong polyanion? High sulfation (~2.5 sulfates/disaccharide).
4. What recognition proteins bind specific sugars on leukocytes/endothelium? Selectins (a class of lectins).
5. Gram-positive vs. Gram-negative—one structural difference: Thick peptidoglycan wall vs. thin wall + outer membrane.

Appendix 1: Protein Purification Techniques

1A. Protein Purification Purpose and Considerations

Purpose: To obtain pure protein for characterization.

Key Considerations:

1. Maintain stability (pH, temperature, proteases, surface adsorption, storage).
2. Use an activity assay (enzymatic rate, ELISA, absorbance @ 280 nm).
3. Purify from cell extract via differential solubility and chromatography.

1B. Salting In, Salting Out, and Precipitation

• Low salt (“salting in”) increases solubility by shielding charges.
• High salt (“salting out”) – e.g., (NH₄)₂SO₄ – competes for water, causing precipitation.
• Collect precipitated protein and dialyze to remove salt.

1C. Chromatography Techniques

UV absorbance @ 280 nm monitors protein fractions.

1D. Electrophoresis and Blotting Methods

• PAGE: Separates based on native charge + size.
• SDS-PAGE: Provides uniform charge → separation based on mass only (MW ∝ 1/log mobility).
• Reducing agents: β-mercaptoethanol / DTT break disulfide bonds.
• IEF (Isoelectric Focusing): Separates by isoelectric point (pI).
• 2D-GE (2D Gel Electrophoresis): IEF followed by SDS-PAGE.
• Blots: Southern (DNA), Northern (RNA), Western (protein).
• Capillary electrophoresis: High resolution, fast analysis.

Appendix 2: Protein Sequencing and Evolution

2A. Protein Sequencing Overview and Strategy

• Why sequence? To determine structure–function links, identify mutations, and study evolution.
• Strategy: Break the protein into overlapping fragments → sequence each fragment → assemble the full sequence.

Disrupting Quaternary Structure

• Separate subunits for individual sequencing.
• N-terminal analysis (dansyl chloride or Edman step 1) reveals the number of chains.
• SDS-PAGE can confirm distinct subunits.

Cleavage of Disulfide Bonds

• Reduce with 2-mercaptoethanol, then alkylate with iodoacetate to form CM-Cys (prevents reoxidation).

Fragmenting the Polypeptide Chain

• Use endopeptidases (e.g., trypsin, which cleaves C-side of Lysine/Arginine unless Proline follows).
• Generate overlapping fragments for assembly.

Edman Degradation Sequencing

1. PITC (Edman reagent) binds the N-terminus, forming a PTC adduct.
2. Trifluoroacetic acid cleaves the first amino acid as a thiazolinone.
3. Convert to PTH-aa → identify by chromatography.
   Repeats cycle-by-cycle for up to 100 amino acids.

Mass Spectrometry (MS/MS) Sequencing

• MS/MS determines peptide mass differences to deduce the sequence order.
• Requires only picomoles of sample.

2F. Protein Sequences and Molecular Evolution

• Cytochrome c: A classic comparison protein; highly conserved across >100 species.
• Invariant residues: Amino acids essential for function that cannot be changed.
• Conservative substitutions: Changes that maintain the chemical character of the residue.
• Hypervariable residues: Residues that tolerate change, used for calculating evolutionary distance.
• Neutral drift: Non-deleterious mutations accumulate over time.
• Histone H4: The most conserved protein (only 2 differences between plants and mammals).
• Hemoglobin / fibrinopeptides: Evolve rapidly.
• Highly expressed proteins = slow evolution (due to folding constraints).
• Phylogenetic trees: Built from sequence similarities to show common ancestry and mutation rates.