Comprehensive Notes on Proteins: Structure, Function, Motifs, and Prions

Structural Levels of Proteins

DNA vs Proteins analogy
- DNA = software, Proteins = hardware
- Nucleotides vs amino acids as building blocks
- Amino acids: 20 common building blocks
- R groups vary in size, shape, charge, polarity, and water solubility
- 20 common amino acids and their symbols
Amino Acids: Building Blocks
- General structure of amino acids
- 20 common amino acids with diverse side chains (R groups)
Peptide Bond
- Formed by dehydration synthesis
- Allows rotation around the attached atoms, enabling various shapes of the polypeptide
Polypeptide Chain
- Unbranched chain of amino acids (aa)
- A protein may consist of one or more polypeptide chains

Levels of Protein Structure

Primary (1°) Structure
- Definition: sequence and number of amino acids
- Covalent bonds in a protein
- Protein amino acid sequence determines higher-level structures
- Example reference: Sickle Cell Anemia highlights the consequence of a single amino acid change
Secondary (2°) Structure
- Localized organization of parts of a polypeptide chain
- Stabilized by hydrogen bonds (H-bonds)
- Between two peptide bonds
- Between a peptide bond and a side chain
- Between two side chains
- Examples of H-bond interactions
- a. H-bond between two peptide bonds:
  - backbone NH --- CO group interactions stabilize structure
- b. H-bond between side chains (e.g., Glu and Ser):
  - example interactions depicted between Glu side chain and Ser side chain
- c. H-bond between a peptide bond and a side chain
- Four kinds of 2° structure
- 1. α-helix: spiral; each turn = 3.6 ext{ aa}; commonly observed; often represented as barrels or rods; stabilized by hydrogen bonds between NH and CO groups of residues; disulfide linkages can play a role in some contexts (S–S)
  - Example: α-keratin in hair
- 2. β-pleated sheet: planar segments arranged in a sheet; hydrogen bonds form between neighboring chains within the sheet
  - Represented by arrows; contributes to strength and stability in structural proteins (e.g., silk fibroin)
- 3. β-turn (β-bend): -shaped reversal of the peptide chain; enables compact globular proteins; often small residues (glycine) or proline with built-in bend
  - Common directions include U-shaped turns with glycine and proline
- 4. Collagen helix: important constituent of connective tissue matrix; stabilized by the association of helices to form a right-handed collagen triple helix
  - Collagen features a characteristic triple-helix assembly
- Secondary structure in some proteins (e.g., ribonuclease A) illustrated as folded sheets and helices
Tertiary (3°) Structure
- Definition: the overall conformation of a single polypeptide chain
- Stabilized by hydrophobic interactions and other non-covalent forces
- Highest level of organization for a single polypeptide
- General shapes observed:
- 1) Fibrous proteins: elongated, single-dimension organization (e.g., keratin, collagen)
  - Functions: external protection (hair, feathers, skin, nails), structural support (tendons, cartilage, bone), deeper layers of skin
- 2) Globular proteins: tightly folded into compact 3D structures
  - More complex than fibrous proteins; includes enzymes, globins
  - Distribution of amino acids
  - Exterior: residues exposed to solvent
  - Interior: hydrophobic residues buried away from solvent
  - Both interior and exterior residues include Pro, Ser, Thr, Cys, Gly, etc.
- Stabilizing forces for tertiary structure (summary)
- Hydrogen bonding between R-groups
- Ionic interactions between oppositely charged R groups
- Hydrophobic interactions (R groups avoiding water)
- Covalent cross-linkages (disulfide bridges)
- Examples of stabilizing interactions depicted:
- H-bonding, ionic bonds, hydrophobic interactions, disulfide bridges, and other covalent/ionic interactions
Quaternary (4°) Structure
- Definition: number and relationships of sub-units in a protein
- Example: Functional bacterial CAP (catabolite activator protein) forms a dimer
- Multimeric assemblies where multiple polypeptide chains associate to form a functional unit

General Principles of Protein Folding

2° structure is determined by short-range sequences of R groups
3° structure is conferred by longer-range aspects of the amino acid sequence
Bend direction and angle are determined by the precise location of residues like Pro, Ser, Thr
Under biological conditions, most polypeptide chains fold into a single, highly stable conformation

Whole-Protein Architecture: Protein Classes

Fibrous proteins
- Polypeptide chains arranged around a single dimension
- Examples: Keratin, Collagen
- Functions: external protection, support and shape, structural components of hair, skin, nails, tendons, cartilage, bone
Globular proteins
- Polypeptide chains folded into a compact 3D structure
- Examples: Enzymes, globins
- Amino acid distribution: exterior vs interior residues

Forces Stabilizing Tertiary Structure (Detailed)

1) Hydrogen bonding between R-groups in adjacent loops or regions
2) Ionic interactions between oppositely charged R groups; or between R groups and water or ions
3) Hydrophobic interactions driving burial of nonpolar residues inside
4) Covalent cross-linkages (Disulfide bridges) between cysteine residues
- Example schematic shows disulfide linkages stabilizing the backbone

Multimeric Proteins: Example of Hemagglutinin

Four levels of structure observed in complex multimeric proteins like hemagglutinin
Each subunit (HA1 and HA2) contributes to the overall assembly

Biological Functions of Proteins

Categories across different functional roles:
- Structural and mechanical roles: keratin, collagen, elastin
- Enzymatic roles: enzymes (e.g., proteases, kinases), ribonucleases
- Transport and storage: lipoproteins, ferritin, myoglobin, hemoglobin
- Defense and signaling: antibodies, transcription factors, hormones
- Regulatory and structural roles: actin, tubulin, myosin, various regulatory proteins
- Gluten and storage proteins: gliadin, ovalbumin, casein
- Proteoglycans, fibrinogen, snake venom proteins
- Proteins with multiple conformations and functions: allosteric and regulatory roles

Enzymes, Cofactors, and Active Sites

Apoenzyme + Cofactor form a holoenzyme
- Protein portion: apoenzyme
- Non-protein portion: cofactor
- Prosthetic group (e.g., heme in peroxidase, FAD in SDH) or Coenzyme (e.g., FMN, NAD in decarboxylases)
- Metal ions (e.g., Mg^{2+} in hexokinase) as cofactors
Active Site of an Enzyme
- Substrate binds at the active site
- Formed by amino acid side chains with two principal roles
- Contact residue: attracts and orients the substrate
- Catalytic residue: participates in transient bond formation with substrate; drives catalysis
Substrate Specificity and Catalysis
- Enzymes are highly specific to substrates and reaction types
- Catalytic changes occur during the reaction cycle
Allosteric Enzymes
- Enzymes exist in alternative conformations with multiple binding sites
- Active site binds substrate; effector site binds regulatory molecules
- Binding of effector molecule induces conformational changes that modulate activity
Allosteric Regulation and Feedback Inhibition
- Feedback inhibition: a downstream product inhibits an enzyme early in the pathway
- Regulation of biosynthetic pathways by effector binding
- Reversible modulation of enzymatic activity through allosteric effectors
Protein-Protein, Protein-RNA, Protein-DNA, and Protein-Drug Interactions
- Molecular interactions drive recognition and binding between biomolecules
- Critical for signaling, regulation, gene expression, and pharmacology

Motifs of DNA-binding Proteins (Secondary Structures in DNA-binding domains)

Motif: a recognizable 2° structure topology that forms a functional DNA-binding domain
Common DNA-binding motifs regulating transcription:

1) Zinc Finger

Finger-like projection of ~30 aa
Two main types depending on how zinc is coordinated:
- Cys2-His2 (C2H2) finger
- Cys4 (C4) finger
2° structure: compact with conserved basic residues; binds major groove of DNA
Zinc finger structure features: two β-sheets and one α-helix
Binds DNA in the major groove; zinc stabilizes fold

2) Helix-Turn-Helix (HTH)

Contains two α-helices and a turn; binds specific sequences in the major groove
Stabilized configuration; recognition helix makes base contacts
Structure: two α-helices and one β-turn (sometimes described as a recognition helix)

3) Leucine Zipper (bZIP)

Leucine every seventh residue on one face forms a dimerization interface
Dimerization domain and DNA-binding domain
Amphipathic dimerization domain; DNA-binding domain is basic and rich in lysine/arginine residues
Function: forms homo- or heterodimers to regulate transcription

4) Helix-Loop-Helix (HLH)

Monomer carries a dimerization domain and a DNA-binding domain
Dimerization domain enables dimer formation; DNA-binding domain engages DNA

5) Copper Fist (Cobalt Finger? Note: historically described as Copper Fist)

Fist-like motif formed around Cu ions which interact with cysteine residues on the protein
Knuckles contain basic amino acids that interact with DNA

Prions

Prions: small proteinaceous infectious particles (no nucleic acid)
Cause neurodegenerative diseases in mammals
Term: Prion Diseases (spongiform encephalopathies)
Nucleic-acid-free spongiform encephalopathy diseases
Examples of prion diseases across species (historical and current context):
- Sheep: Scrapie (first discovered by Stanley Prusiner, 1892)
- Transmissible Mink Encephalopathy (TME) in mink
- Chronic Wasting Disease (CWD) in mule deer, elk
- Bovine Spongiform Encephalopathy (BSE) in cows
- Creutzfeldt–Jakob disease (CJD) in humans
- Gerstmann–Straussler–Scheinker syndrome (GSS)
- Fatal Familial Insomnia (FFI)
- Kuru
- Alpers syndrome in infants
Symptoms of Prion Diseases (stages)
- Early stages: loss of muscle control, personality changes, impaired memory/judgment/thinking, impaired vision, insomnia, depression, dementia
- Later stages: involuntary muscle jerks, blindness, paralysis, wasting, coma, death (often following pneumonia)
- Visible end results at autopsy: non-inflammatory lesions, vacuoles, amyloid protein deposits, astrogliosis
Kuru
- First known prion disease (1950s)
- Geographically isolated tribes in the highlands of New Guinea
- Transmission believed to be via ingestion of brain tissue during religious rituals
Molecular Biology of Prions
- Prion protein (PrP) exists in different conformations
- PrP^C (cellular PrP): normal protein; encoded by a single exon of a single-copy gene on chromosome 20; predominantly on neuron surfaces; non-immunogenic and protease-sensitive
- PrP^Sc (scrapie-associated isoform): modified form that is relatively protease-resistant and accumulates in diseased individuals
- The infectious process involves conversion of PrP^C to PrP^Sc; the exact mechanism is unknown; may involve conformational change or chemical modification
Structural Difference Between PrP^C and PrP^Sc
- PrP^C: predominantly α-helix structure
- PrP^Sc: predominantly β-pleated sheet structure
- This conformational shift underlies prion infectivity and aggregation tendencies
Normal cellular PrP (PrP^C) and its chromosomal locus
- PrP^C encoded on chromosome 20; normal cellular form associated with synaptic function
- PrP^Sc accumulates in synaptic vesicles and has a different conformational profile
Conceptual Summary
- Prions are protein-only infectious agents that propagate by converting normal PrP to the misfolded PrP^Sc form
- Conformational changes alter properties and lead to disease progression