13.4 Amino Acids & Proteins: Structure, Properties, Separation, Specialized Products, Collagen & Protein Misfolding in Alzheimer Disease

Why this area matters

The TOS lists "amino acid and protein biochemistry (structure, function, properties, denaturation, fractionation, chemical reactions, separation), collagen, Alzheimer's disease". Expect items on:

• the charge of amino acids at a given pH;
• what denaturation destroys;
• reading a serum protein electrophoresis;
• matching a collagen defect to its disease;
• the molecular basis of Alzheimer disease.

Hierarchy of Protein Architecture and Conformational Dynamics

Proteins fold into thermodynamically stable native conformations structured across four discrete organizational tiers:

1. Primary Structure: The linear sequence of amino acids linked covalently by peptide bonds (amide linkages connecting the $\alpha$-carboxyl group of one amino acid to the $\alpha$-amino group of the next). Peptide bonds possess partial double-bond character (40% resonance stabilization) that restricts rotation, maintaining a rigid, planar trans configuration.
2. Secondary Structure: Local spatial arrangements of the polypeptide backbone stabilized strictly by hydrogen bonds between carbonyl oxygens ($C=O$) and amide nitrogens ($N-H$):
   • $\alpha$-Helix: Right-handed spiral with $3.6$ amino acids per turn. Hydrogen bonds form between the $C=O$ of residue $n$ and the $N-H$ of residue $n+4$. Proline acts as an "$\alpha$-helix breaker" because its rigid cyclic pyrrolidine ring introduces a steric kink and lacks a free amide hydrogen for hydrogen bonding. Glycine can also disrupt helices due to excessive conformational flexibility.
   • $\beta$-Pleated Sheet: Extended parallel or antiparallel polypeptide strands stabilized by interchain hydrogen bonds perpendicular to the polypeptide backbone.
3. Tertiary Structure: The overall three-dimensional folding of a single polypeptide chain into domain structures. Stabilized by hydrophobic interactions (burying nonpolar side chains in the core), electrostatic salt bridges (between positively charged Lys/Arg/His and negatively charged Asp/Glu), hydrogen bonds, and covalent disulfide bonds formed between cysteine sulfhydryl groups via oxidation.
4. Quaternary Structure: The spatial assembly of two or more independent polypeptide subunits into a multimeric functional complex (e.g., adult hemoglobin $\alpha_2\beta_2$ heterotetramer).

Molecular Chaperones, Misfolding Pathologies, and Amyloidosis

Proper conformational folding is guided by molecular chaperones, including Heat Shock Proteins (HSP70, HSP60), which bind exposed hydrophobic segments of nascent polypeptides to prevent inappropriate aggregation. When protein quality control fails, misfolded proteins aggregate into insoluble fibrillar structures dominated by antiparallel cross-$\beta$-pleated sheets, termed amyloid.

Amyloid deposits resist endogenous proteolysis and disrupt organ function. Histologically, amyloid binds Congo red dye, demonstrating pathognomonic apple-green birefringence under polarized light microscopy:

• AL Amyloidosis (Primary): Deposition of monoclonal immunoglobulin light chains ($\kappa$ or $\lambda$) produced in plasma cell dyscrasias (multiple myeloma).
• AA Amyloidosis (Secondary): Deposition of Serum Amyloid A (SAA), an acute-phase reactant synthesized in the liver during chronic inflammatory states (tuberculosis, bronchiectasis, rheumatoid arthritis).
• $A\beta$ Amyloidosis: Deposition of $\beta$-amyloid cleaved from Amyloid Precursor Protein (APP) on chromosome 21, forming senile plaques in Alzheimer disease and Down syndrome.
• $\beta_2$-Microglobulin Amyloidosis: Accumulation of MHC Class I light chains in patients undergoing long-term hemodialysis, causing carpal tunnel syndrome and destructive spondyloarthropathy.

---

Amino Acid Nutritional and Metabolic Classification

All standard 20 amino acids feature a chiral $\alpha$-carbon (except glycine). They are categorized according to nutritional essentiality and the metabolic destinations of their carbon skeletons:

Essential versus Non-Essential Amino Acids

Nine amino acids cannot be synthesized de novo in humans and must be supplied in the diet:

• Mnemonic: "PVT TIM HaLL" (Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Leucine, Lysine).

Glucogenic versus Ketogenic Amino Acids

Net Charge at Physiologic pH (7.4)

• Acidic Amino Acids (Negatively Charged): Aspartate ($\text{p}K_a \approx 3.9$) and Glutamate ($\text{p}K_a \approx 4.1$).
• Basic Amino Acids (Positively Charged): Lysine ($\text{p}K_a \approx 10.5$), Arginine ($\text{p}K_a \approx 12.5$, the most basic amino acid), and Histidine ($\text{p}K_a \approx 6.0$). Because histidine's imidazole side chain has a $\text{p}K_a$ near physiological pH, it can rapidly exchange protons, making it a critical catalytic residue in enzyme active sites and the primary protein buffer in hemoglobin.

Physical properties, denaturation and separation

• Isoelectric point (pI): the pH at which a molecule carries no net charge.
  • Below its pI, a protein is positively charged and moves to the cathode; above its pI it is negative and moves to the anode.
  • At physiologic pH (7.4), most plasma proteins are negatively charged, because their pI is about 5 to 6.
  • Solubility is lowest at the pI, which is used in isoelectric precipitation.
• Denaturation: loss of secondary, tertiary and quaternary structure, with peptide bonds intact, so the primary structure survives. It is caused by heat, extreme pH, organic solvents, urea, detergents and heavy metals. Biological activity is lost. It is sometimes reversible (ribonuclease refolding), but usually not, as when egg white is cooked. Denatured proteins are more easily digested, which is one reason cooking helps.
• Chemical reactions and assays:
  • Biuret (peptide bonds) and Lowry methods measure total protein.
  • Ninhydrin detects free amino groups.
  • Sanger and Edman reagents identify N-terminal residues.
• Fractionation and separation:

Serum protein electrophoresis (anode to cathode):

1. Albumin: the largest band. It falls in liver disease, nephrotic syndrome and malnutrition.
2. Alpha-1: alpha-1-antitrypsin. It is low in AAT deficiency.
3. Alpha-2: haptoglobin, alpha-2-macroglobulin, ceruloplasmin. It rises in inflammation and nephrotic syndrome.
4. Beta: transferrin, C3, LDL. Transferrin rises in iron deficiency.
5. Gamma: immunoglobulins.
   • A narrow monoclonal (M) spike suggests myeloma or MGUS.
   • A broad polyclonal increase suggests chronic infection, autoimmunity or cirrhosis; cirrhosis also shows beta-gamma bridging from IgA.
   • Low gamma suggests agammaglobulinemia.

Connective Tissue Architecture & Collagen Biochemistry

Connective tissue consists of cells (fibroblasts, adipocytes, macrophages, mast cells) suspended within an abundant extracellular matrix (ECM) composed of ground substance (glycosaminoglycans like hyaluronic acid, chondroitin sulfate; proteoglycans; multiadhesive glycoproteins like fibronectin and laminin) and structural fibers.

The Major Collagen Subtypes

Collagen is the most abundant protein in the human body, synthesized as procollagen triple helices containing repeating Gly-X-Y amino acid sequences (where X is typically proline and Y is hydroxyproline or hydroxylysine). Intracellular hydroxylation of proline and lysine requires ascorbic acid (vitamin C); failure of this step leads to impaired triple-helix hydrogen bonding and clinical scurvy (poor wound healing, gum bleeding, perifollicular hemorrhages).

1. Type I Collagen:

   • Distribution: 90% of total bodily collagen. Present in bone (organic osteoid), skin dermis, tendons, ligaments, fascia, cornea, dentin, and mature scar tissue during late wound remodeling ($> 3\text{ weeks}$ post-injury).
   • Clinical Pathology: Mutational defects in COL1A1 or COL1A2 genes cause Osteogenesis Imperfecta (autosomal dominant; recurrent pathological fractures with minimal trauma, blue sclerae due to translucent choroidal visualization, hearing loss from stapes footplate ankylosis, and opalescent teeth in dentinogenesis imperfecta).

2. Type II Collagen:

   • Distribution: Chief fibrillar constituent of hyaline cartilage, elastic cartilage, the vitreous body of the eye, and the nucleus pulposus of the intervertebral disc ("car-two-lage").
   • Function: Resists intermittent compressive loads in articular joints.

3. Type III Collagen (Reticulin):

   • Distribution: Forms delicate, pliable argyrophilic reticular fiber meshes in highly distensible, cellular organs: blood vessel walls (aorta, muscular arteries), dermis, uterus, lymphoid organs (spleen, lymph nodes), and early granulation tissue (synthesized rapidly during early wound repair before enzymatic replacement by Type I collagen).
   • Clinical Pathology: Mutations in COL3A1 cause Vascular Ehlers-Danlos Syndrome (Type IV EDS; arterial aneurysm rupture, spontaneous bowel perforation, and gravid uterine rupture due to vascular fragility).

4. Type IV Collagen:

   • Distribution: Forms the non-fibrillar, two-dimensional sheet meshwork of the basal lamina (lamina densa) underlying all epithelia, surrounding muscle cells, and forming the renal glomerular basement membrane (GBM) ("Type four on the floor").
   • Clinical Pathology:
     • Alport Syndrome: X-linked defect in the $\alpha_5$ chain of Type IV collagen (COL4A5), manifesting as progressive hereditary nephritis with microhematuria, sensorineural hearing loss, and ocular abnormalities (anterior lenticonus).
     • Goodpasture Syndrome: Autoantibodies directed against the non-collagenous NC1 domain of the $\alpha_3$ chain of Type IV collagen, causing rapidly progressive glomerulonephritis and pulmonary alveolar hemorrhage with linear IgG immunofluorescence.

Collagen synthesis step by step

1. Translation of pre-procollagen alpha chains on the rough ER.
2. Hydroxylation of proline and lysine by prolyl and lysyl hydroxylases. These need vitamin C, Fe2+, O2 and alpha-ketoglutarate; vitamin C deficiency causes scurvy.
3. Glycosylation of hydroxylysine.
4. Assembly of the procollagen triple helix, stabilized by hydrogen bonds. Osteogenesis imperfecta mutations disrupt this; glycine substitutions are the most severe.
5. Secretion into the extracellular space and cleavage of the terminal propeptides. Defective cleavage causes some forms of Ehlers-Danlos syndrome.
6. Cross-linking by lysyl oxidase, a copper-dependent enzyme. Menkes disease (an X-linked copper-transport defect, ATP7A) causes brittle "kinky" hair, hypotonia and arterial fragility.

Elastin, rich in glycine and proline but not hydroxylated, is cross-linked through desmosine. Fibrillin-1 is its scaffold, and Marfan syndrome is caused by fibrillin-1 mutations.

Specialized Products Derived from Amino Acids

Protein misfolding and Alzheimer disease

• Amyloid-beta: amyloid precursor protein (APP, chromosome 21) is normally cut by alpha-secretase. Sequential cleavage by beta-secretase and gamma-secretase (presenilins) instead releases amyloid-beta 42, which aggregates into extracellular neuritic plaques and vessel-wall amyloid.
• Tau: hyperphosphorylated tau, a microtubule-associated protein, forms intracellular neurofibrillary tangles, which correlate best with dementia severity.
• Genetic risk:
  • early-onset familial disease: mutations of APP, PSEN1 or PSEN2; trisomy 21 adds an APP copy;
  • late-onset disease: APOE e4 raises risk, and APOE e2 is protective.
• Neurochemistry: loss of cholinergic neurons in the nucleus basalis of Meynert lowers acetylcholine.
  • Acetylcholinesterase inhibitors (donepezil, rivastigmine, galantamine) give modest symptomatic benefit.
  • Memantine (an NMDA antagonist) limits glutamate excitotoxicity.
  • Anti-amyloid antibodies remove plaque but carry a risk of brain swelling and hemorrhage (ARIA).
• Other misfolding diseases:
  • prion diseases (Creutzfeldt-Jakob: PrP-sc converts normal prion protein);
  • Parkinson disease (alpha-synuclein);
  • Huntington disease (polyglutamine);
  • alpha-1-antitrypsin deficiency (misfolded protein trapped in hepatocytes).