04 Proteins

Table of Contents

1. Introduction to Proteins
2. Protein Functions
3. Protein Synthesis
4. Levels of Protein Structure
5. Primary Structure
6. Protein Folding: Thermodynamic Principles
7. Secondary Structure
   • α-Helix
   • β-Pleated Sheets
   • β-Turns
   • Random Coil
8. Tertiary Structure
9. Quaternary Structure
10. Fibrous Proteins
    • α-Keratins
    • Collagen
11. Globular Proteins
12. Protein Folding, Denaturation, and Proteostasis
13. Post-Translational Modifications
14. Conjugated Proteins and Prosthetic Groups
15. Classification of Proteins
16. Membrane Proteins
17. Summary and Clinical Connections

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1. Introduction to Proteins

Proteins are the most functionally diverse class of biological macromolecules. They are linear polymers made up of amino acids connected end-to-end by peptide bonds, forming polypeptide chains. These chains then fold into precise three-dimensional shapes that determine protein function.

Key introductory facts:

• Not all amino acids are equally abundant in proteins. Leucine is the most common, while tryptophan and cysteine are relatively rare.
• Some proteins consist of more than one polypeptide chain (called subunits). These are called oligomeric proteins. The subunits may be identical or different (e.g., hemoglobin has two α and two β chains).
• The three-dimensional (3D) shape a protein adopts in its functional state is called its native structure. This shape is critical for its biological activity.

Context: Proteins are distinct from the other macromolecules (carbohydrates, lipids, nucleic acids) in that their function is almost entirely determined by their 3D shape, which in turn is determined by their amino acid sequence.

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2. Protein Functions

Proteins perform an extraordinary range of biological functions, all intimately related to their chemical structure. Key functional categories include:

Function	Description	Example
Catalysts (Enzymes)	Accelerate chemical reactions	RUBISCO (carbon fixation)
Structural	Provide mechanical support	Collagen, Spider silk
Transport	Carry molecules through blood or membranes	Hemoglobin (O₂), Albumin
Storage	Store nutrients or metal ions	Ferritin (iron storage)
Messengers (Hormones)	Signal between cells and organs	Insulin
Antibodies (Immunoglobulins)	Recognize and neutralize foreign agents	IgG
Regulatory (Receptors)	Receive and transduce signals	Rhodopsin
Contractile/Motor	Enable movement	Actin and Myosin
Toxic	Defense or predation	Various toxins

Key principle: The function of a protein is inseparable from its structure. Disrupting the shape of a protein typically abolishes its function.

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3. Protein Synthesis

Proteins are synthesized on ribosomes in the cytosol. The process flows from DNA to protein in a two-stage sequence:

1. Transcription: A segment of DNA (a gene) is copied into messenger RNA (mRNA) in the nucleus.
2. Translation: The mRNA sequence is read at the ribosome. Transfer RNA (tRNA) molecules bring the correct amino acids, and the ribosome links them together in the order dictated by the mRNA codons.

The genetic code is the set of rules by which nucleotide triplets (codons) in the mRNA are interpreted as specific amino acids. This information flow — DNA → mRNA → protein — is called the Central Dogma of Molecular Biology.

Visualization: Imagine the ribosome as a factory reading a blueprint (mRNA) and assembling a product (protein) from parts (amino acids) delivered by tRNA molecules.

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4. Levels of Protein Structure

To describe the complex architecture of proteins, four hierarchical levels of organization are used:

Level	Description
Primary	The linear sequence of amino acids in the polypeptide chain
Secondary	Local, regular folding patterns stabilized by hydrogen bonds (e.g., α-helix, β-sheet)
Tertiary	The overall 3D folding of a single polypeptide chain
Quaternary	The association of two or more polypeptide chains into a multi-subunit complex

Each level builds on the one before. Ultimately, the primary structure (amino acid sequence) contains all the information needed to specify the higher levels of structure — a concept proven by Anfinsen’s renaturation experiments (see Section 12).

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5. Primary Structure

Definition

The primary structure is the sequence of amino acids in the polypeptide chain, linked together by peptide bonds. Amino acids are connected in a “head-to-tail” fashion: the carboxyl group (–COOH) of one amino acid reacts with the amino group (–NH₂) of the next, releasing water (a condensation reaction).

The Peptide Bond

The peptide bond (C–N bond between residues) has a critical structural feature: it has partial double-bond characterdue to resonance delocalization of electrons between the nitrogen lone pair and the adjacent carbonyl (C=O). This means:

• The peptide bond cannot rotate freely — it is rigid and planar.
• The atoms around the peptide bond are locked in either a trans or cis configuration (>90% of peptide bonds are trans).

However, the bonds flanking the central Cα (alpha carbon) can rotate:

• Φ (phi): the bond between N–Cα
• Ψ (psi): the bond between Cα–C

These rotations (within the steric constraints imposed by the R-groups) are what allow a polypeptide chain to fold.

The Backbone and Side Chains

The repeating units of N–Cα–C form the backbone of the protein. The variable R groups (side chains) attached to each Cα project outward from the backbone and are responsible for the chemical diversity and folding of proteins.

• The chain always begins with a free amino group (N-terminus) and ends with a free carboxyl group (C-terminus).
• By convention, the sequence is always read from N-terminus to C-terminus.

Importance of Sequence: Sickle Cell Anemia

The primary structure dictates everything. A change of even a single amino acid can have dramatic functional consequences.

Example — Sickle Cell Anemia:

• Normal hemoglobin (HbA) contains glutamate (Glu) — a polar, negatively charged amino acid — at position 6 of the β chains.
• In sickle cell hemoglobin (HbS), this is replaced by valine (Val) — a nonpolar, aliphatic amino acid.
• When HbS is deoxygenated, it becomes insoluble and polymerizes into long, rigid fibers. These distort red blood cells into a sickle shape, blocking capillaries and causing pain, organ damage, and early death.

Clinical relevance: Sickle cell anemia is a classic example of a molecular disease — a disorder caused by a single point mutation altering one amino acid in a protein.

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6. Protein Folding: Thermodynamic Principles

Why Do Proteins Fold?

Protein folding is driven by thermodynamics. Proteins adopt the native conformation that has the lowest Gibbs free energy (G). The relevant equation is:

$\Delta G=\Delta H-T\Delta S$

For folding to be spontaneous, ΔG must be negative.

Components of the Folding Energy

Enthalpy (ΔH):

• Favorable (negative ΔH): Noncovalent intramolecular forces — hydrogen bonds and van der Waals interactions — form as the protein folds, releasing energy.

Entropy (ΔS) — two opposing contributions:

• Unfavorable (negative ΔS): The polypeptide chain loses conformational freedom as it folds → decrease in conformational entropy.
• Favorable (positive ΔS): The hydrophobic effect — nonpolar side chains cluster together in the protein core, releasing ordered water molecules from the solvation shell around them into the bulk solvent. This increases the entropy of the water, providing a net favorable entropic contribution.

The Hydrophobic Effect (in detail)

Nonpolar (hydrophobic) amino acid side chains are poorly compatible with water. In an unfolded protein, water molecules form an ordered “cage” (clathrate-like structure) around hydrophobic groups to minimize unfavorable interactions. This ordering of water decreases entropy.

When the protein folds and buries hydrophobic residues in the interior core:

• The ordered water molecules are released into the bulk solvent → large increase in water entropy.
• This is the major thermodynamic driving force for folding.

Summary of Stabilizing Interactions

Interaction	Type	Role
Hydrophobic effect	Entropic	Major driving force; buries nonpolar residues in core
Hydrogen bonds	Enthalpic	Stabilize α-helices and β-sheets
London dispersion (van der Waals)	Enthalpic	Weak but numerous; stabilize the protein interior
Electrostatic interactions (salt bridges)	Enthalpic	Long-range; strong when buried in hydrophobic environment
Disulfide bonds	Covalent	Stabilize tertiary structure (mainly in extracellular proteins)

Net result: The favorable enthalpy from noncovalent interactions and the favorable entropic contribution of the hydrophobic effect outweigh the unfavorable conformational entropy loss, giving a net negative ΔG for folding.

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7. Secondary Structure

Secondary structure refers to local, regular spatial arrangements of the polypeptide backbone, stabilized primarily by hydrogen bonds between the carbonyl oxygen (C=O) of one peptide bond and the amide hydrogen (N–H) of another. The R-groups do not participate in defining secondary structure.

The main types are: α-helix, β-pleated sheet, and β-turn.

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7.1 The α-Helix

First described by Linus Pauling and Robert Corey in 1951 (Pauling received the Nobel Prize in Chemistry in 1954).

Structure

• The polypeptide backbone coils into a right-handed helix (clockwise when viewed from the N-terminal end).
• There are 3.6 amino acid residues per turn.
• The pitch (rise per turn) is 5.4 Å (0.54 nm).
• Each C=O group forms a hydrogen bond with the N–H of the amino acid 4 residues further along the chain (i→i+4 hydrogen bonding pattern).
• All R-groups point outward from the helix axis, away from the backbone.

Visualization: Think of the helix like a coiled telephone cord — the backbone forms the cord, the R-groups stick outward like bristles.

Handedness

• Only right-handed α-helices are found in proteins (with very rare exceptions).
• Left-handed α-helices are theoretically possible but are sterically unfavorable for L-amino acids and are extremely rare (31 verified in 7,284 proteins studied).

Helix Stability: Sequence Effects

Not all amino acids promote α-helix formation equally:

Helix promoters:

• Alanine (Ala): Highest intrinsic helix-forming propensity of all amino acids; small, methyl side chain causes minimal steric clash.
• Leucine (Leu): Also a good helix former.

Helix breakers:

• Proline (Pro): The nitrogen of proline is part of a rigid ring structure (imino nitrogen), meaning there is no N–H available for hydrogen bonding and rotation around the N–Cα bond is impossible. Pro causes a kink or break in the helix.
• Glycine (Gly): The absence of a side chain (R = H) makes Gly too flexible and allows conformations incompatible with the regular α-helix. Gly better supports left-handed helices.
• Charged amino acids in runs (e.g., Glu–Glu–Glu or Lys–Lys–Lys): Repulsion between like charges destabilizes the helix.
• Bulky or branched R-groups adjacent to each other can sterically clash.

α-Helices in Membrane Proteins

Proteins that span the lipid bilayer (transmembrane proteins) use α-helices as their transmembrane segments. These helices are composed predominantly of nonpolar (hydrophobic) amino acids, whose R-groups point outward and interact favorably with the hydrophobic core of the membrane. Membrane proteins can cross the bilayer once (single-pass), multiple times (multi-pass), or as a β-barrel (a rolled-up β-sheet).

Amphipathic α-helices have a nonpolar face and a polar face, allowing them to associate with the membrane surface on the cytosolic side.

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7.2 β-Pleated Sheets

Structure

• The β-sheet is formed by β-strands — extended segments of the polypeptide chain.
• In a β-strand, the backbone is nearly fully extended; R-groups alternate above and below the plane of the strand, creating a zigzag pattern.
• Hydrogen bonds form between adjacent β-strands (inter-strand), not within a single strand.
• Multiple β-strands align side-by-side to form the sheet.

Parallel vs. Antiparallel

Type	Orientation of strands	Hydrogen bonds	Stability
Antiparallel	N→C runs opposite to adjacent strand	Nearly in-line (optimal geometry)	More stable
Parallel	N→C runs same direction	Distorted, not in-line	Less stable

Note: β-strands forming a single β-sheet can be nearby or far apart in the primary sequence, and they can even come from different polypeptide chains.

Preferred Amino Acids

• Small R-groups (e.g., Gly) are preferred when β-sheets are stacked in layers, to avoid steric clashes between the sheets.
• Destabilizing factors: Large bulky R-groups; electrostatic repulsion or attraction between charged R-groups in adjacent strands.

Example — Silk Fibroin

• Fibroin (the protein of silk) has the highest β-sheet content of any known protein, due to long stretches of Gly-Ala-Gly-Ala sequence.
• Alternating Gly (R = H, small) and Ala (R = CH₃, small) allows the sheets to pack tightly together.
• This gives silk its characteristic toughness and rigidity, while the regions between the β-sheets provide some flexibility.

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7.3 β-Turns

β-turns (also called reverse turns or hairpin turns) are compact structural elements that allow the polypeptide chain to change direction sharply — typically reversing course by 180°.

Structure

• Involves 4 consecutive amino acid residues.
• A hydrogen bond forms between the carbonyl oxygen of residue 1 and the amide hydrogen of residue 4.
• The central two residues (positions 2 and 3) do not participate in inter-residue hydrogen bonding.
• β-turns are frequently found near the surface of a protein, where the central residues can hydrogen-bond with water.

Amino Acids Favored in β-Turns

• Glycine (Gly): Small and flexible — can adopt unusual backbone angles.
• Proline (Pro): The rigid ring of proline naturally constrains the backbone to an angle compatible with a tight turn. Note: about 6% of Pro peptide bonds are in the cis configuration, facilitating β-turns.

β-turns typically connect the ends of two adjacent antiparallel β-strands and are essential architectural features of globular proteins.

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7.4 Random Coil

Regions of the polypeptide that do not adopt a regular, repetitive secondary structure are referred to as random coils (or loops/linkers).

Features:

• 4–20 amino acid residues in length (non-repetitive).
• No consistent hydrogen bonding pattern between backbone atoms.
• Found as terminal arms (both N-terminus and C-terminus) and as loops connecting regular secondary structure elements (α-helices or β-sheets).

Important modern concept: Some proteins, called intrinsically disordered proteins (IDPs), have large regions that are permanently unstructured under physiological conditions. These disordered regions often become structured when they bind their target molecule, enabling highly specific, rapidly regulated interactions. This is a major area of current protein science research.

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8. Tertiary Structure

Definition

Tertiary structure describes the complete three-dimensional folding of a single polypeptide chain — the overall arrangement of all its atoms in space, including all secondary structures, loops, and turns.

While secondary structure describes local arrangements, tertiary structure describes long-range interactions: amino acids that are far apart in the primary sequence can come into close proximity in the folded protein.

Stabilizing Interactions

The tertiary structure is maintained by a combination of:

1. Hydrophobic effect: Nonpolar side chains are buried in the hydrophobic core, away from the aqueous environment.
2. Hydrogen bonds: Between polar side chains and between the backbone groups not already engaged in secondary structures.
3. Electrostatic (ionic) interactions: Salt bridges between oppositely charged side chains (e.g., –NH₃⁺ of Lys and –COO⁻ of Asp).
4. Van der Waals forces: Weak, short-range attractions between all atoms that are in close proximity.
5. Disulfide bonds (–S–S–): Covalent bonds formed by the oxidation of two cysteine –SH (thiol) groups that are spatially adjacent.

Disulfide Bonds

• The formation of a disulfide bond requires an oxidizing environment.
• Inside cells, the cytoplasm is highly reducing (due to glutathione and other reductants) → most intracellular proteins do not have disulfide bonds.
• In eukaryotes, disulfide bonds are primarily found in secreted, extracellular proteins (e.g., insulin, immunoglobulins) and proteins in the endoplasmic reticulum lumen.

Glutathione (γ-Glu–Cys–Gly) is the major intracellular thiol buffer, keeping cysteine residues reduced in the cytoplasm.

Interior vs. Exterior Arrangement

• The interior of a globular protein is predominantly nonpolar (hydrophobic side chains: Val, Leu, Phe, Ile).
• The exterior (surface) is predominantly polar and charged (hydrophilic side chains: Lys, Asp, Glu, Ser) — enabling interaction with the aqueous environment.

Structural Domains and Motifs

• A motif (fold): A recognizable, recurring folding pattern involving different secondary structures and their connections (e.g., the β-barrel, the coiled-coil).
• A domain: An independently stable region of the polypeptide that can fold and function on its own. Larger proteins (>200–300 amino acids) often have multiple domains, each with distinct functions (e.g., a DNA-binding domain, a catalytic domain).

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9. Quaternary Structure

Definition

Quaternary structure applies only to proteins that consist of two or more polypeptide chains (subunits). It describes the arrangement and interactions between those subunits.

Stabilizing Interactions

Subunits are held together by the same non-covalent forces that stabilize tertiary structure:

• Electrostatic attractions
• Hydrogen bonds
• Van der Waals forces
• Occasionally, disulfide bridges between chains

Terminology

Term	Meaning
Monomer	Single polypeptide chain
Dimer	Two subunits
Trimer	Three subunits
Tetramer	Four subunits
Homodimer/Homotrimer…	All subunits identical
Heterodimer/Heterotrimer…	Subunits are different
Protomer	The identical repeating unit in an oligomeric protein

Example: Hemoglobin

• Hemoglobin is a heterotetramer: 2 α chains + 2 β chains (α₂β₂).
• All four subunits are held together by noncovalent interactions.
• It can also be viewed as a dimer of αβ protomers.
• Each subunit carries a heme prosthetic group (iron-containing porphyrin ring) that binds O₂.

Advantages of Quaternary Association

1. Stability: Reduces the surface-to-volume ratio; buried interfaces are protected from water.
2. Genetic economy: One gene can encode a subunit used multiple times.
3. Bringing catalytic sites together: Multi-enzyme complexes carry out sequential reactions efficiently.
4. Cooperativity: Binding of a ligand to one subunit can influence the others (allosteric regulation) — critical for oxygen binding in hemoglobin.

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10. Fibrous Proteins

Fibrous proteins are characterized by:

• One predominant type of secondary structure throughout.
• Extended, filamentous or sheet-like shapes.
• Insolubility in water (due to high content of hydrophobic amino acids on their surfaces, which pack tightly together in supramolecular bundles).
• Structural or protective functions in tissues.

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10.1 α-Keratins

Overview

• α-Keratins are the major proteins of hair, wool, nails, horns, hooves, and the outer layer of skin in mammals.
• They consist almost entirely of α-helical secondary structure.
• Insoluble, not very reactive, rich in hydrophobic amino acids, and organized into long filaments bundled together.

Hierarchical Structure (from small to large)

1. Single α-helix (right-handed).
2. Coiled-coil dimer: Two α-helices wrap around each other in a left-handed supertwist, forming a protofilament(stabilized largely by disulfide bonds between cysteine residues).
3. Protofilament: Two coiled-coil dimers associate.
4. Protofibril: Two protofilaments.
5. Intermediate Filament: Four protofibrils polymerize to form the ~10 nm diameter intermediate filament, the basic subunit of α-keratin fibers.

Visualization: Think of it like a multi-strand rope — individual helices twist together like strands, which in turn twist together to form the final thick cable.

Hard vs. Soft α-Keratins

The mechanical properties of α-keratins depend on their cysteine content and hence the number of disulfide bonds:

Type	Disulfide Bond Density	Examples	Properties
Soft α-keratin	Low	Hair, skin	Flexible, pliable
Hard α-keratin	High	Nails, horns, hooves	Rigid, hard, resistant to force

The Permanent Wave (Biochemical Application)

A permanent wave exploits the chemistry of disulfide bonds:

1. Reduction: A reducing agent breaks the disulfide bonds (–S–S– → 2 –SH), allowing the keratin chains to move freely.
2. Reshaping: The hair is wrapped around curlers to impose a new shape.
3. Oxidation: An oxidizing agent is applied, reforming disulfide bonds in the new positions dictated by the curlers.
4. Result: The hair retains its new curved shape because the disulfide crosslinks now lock it into the new conformation.

The Coiled-Coil: A Supersecondary Structure

The coiled-coil motif (2–7 α-helices wound together like rope strands) is not limited to keratins. It is found in many proteins involved in:

• Gene regulation (e.g., transcription factors with leucine zipper motifs).
• Cytoskeletal proteins (intermediate filaments).
• Viral fusion proteins and many others.

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10.2 Collagen

Overview

• Collagen is the most abundant protein in the human body, comprising 25–35% of total body protein.
• It is a major component of skin, tendons, cartilage, ligaments, bone matrix, and the cornea.
• Its function is to provide structural integrity and tensile strength to connective tissues.

Amino Acid Composition

• 35% Glycine (Gly)
• 11% Alanine (Ala)
• 21% Proline (Pro) and Hydroxyproline (4-Hyp)

The repeating sequence motif is: Gly – X – Y, where:

• X is often Proline (Pro)
• Y is often 4-Hydroxyproline (4-Hyp) or Hydroxylysine (HyLys)

Unique Secondary Structure: The Collagen Helix

The collagen helix is completely distinct from the α-helix:

• It is left-handed (opposite handedness to the α-helix).
• It has only 3 amino acid residues per turn (much more extended than the α-helix with 3.6).
• Glycine must occupy every third position (Gly-X-Y) because glycine — with R = H — is the only amino acid small enough to fit into the sterically constrained center of the triple helix.

Tertiary/Quaternary Structure: The Triple Helix

Three collagen α-chains (each a left-handed collagen helix) wrap around each other in a right-handed superhelix, forming the collagen triple helix (tropocollagen molecule).

• Diameter: ~1.4 nm
• The three chains are staggered by one residue.
• The tight wrapping provides extraordinary tensile strength — collagen has higher tensile strength than steel wire of the same cross-section.

Post-translational Modifications in Collagen

Collagen is a glycoprotein: it undergoes O-linked glycosylation on hydroxylysine residues (attachment of glucose and galactose).

The hydroxylation of Pro and Lys requires vitamin C (ascorbate) as a cofactor for the enzymes prolyl hydroxylase and lysyl hydroxylase.

Scurvy: A Collagen Disease

Scurvy results from vitamin C deficiency.

• Without vitamin C, prolyl hydroxylase cannot function → proline residues cannot be hydroxylated → 4-hydroxyproline is not produced.
• Without hydroxyproline, the collagen triple helix cannot form properly and cannot be crosslinked.
• Consequence: Collagen fibers lose their tensile strength.
• Clinical features: Fragile blood vessels (capillary fragility → bruising and bleeding), poor wound healing, loosening of teeth, bleeding gums, joint pain.
• Historically fatal in sailors on long voyages without fresh fruit/vegetables.

Historical quote from The Memoirs of the Lord of Joinville, ca. 1300: “The signal was this: when the nose began to bleed, then death was at hand.”

Hierarchical Assembly of Collagen

1. Amino acid sequence: Gly–X–Y repeats
2. α-chains: Individual left-handed collagen helices
3. Tropocollagen molecule: Three α-chains form the right-handed triple helix (~300 nm long)
4. Collagen fibrils: Tropocollagen molecules pack together in a staggered arrangement
5. Collagen fibers: Multiple fibrils bundled together
6. Fiber bundles/Fascicles: Final macroscopic collagen structure

Comparison to α-Keratin

Feature	α-Keratin	Collagen
Secondary structure	α-helix (right-handed)	Collagen helix (left-handed)
Superstructure	Left-handed coiled-coil	Right-handed triple helix
Key amino acids	Cys (disulfide bonds), hydrophobic AAs	Gly (every 3rd), Pro, Hyp, HyLys
Stabilizing bonds	Disulfide bonds	Hydrogen bonds, crosslinks
Function	Hair, nails, skin protection	Connective tissue strength

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11. Globular Proteins

In contrast to fibrous proteins, globular proteins have:

• Multiple types of secondary structure (α-helices, β-sheets, loops — all within one protein).
• A compact, roughly spherical shape achieved by extensive folding back of the polypeptide.
• Hydrophobic core and hydrophilic surface (same principle as in any folded protein).
• Solubility in water (because the exterior is hydrophilic).
• Diverse biological functions: enzymes, antibodies, carrier proteins, hormones, regulatory proteins.

Examples

• Enzymes: e.g., Ribonuclease A (digests RNA), RUBISCO (CO₂ fixation in plants).
• Antibodies (Immunoglobulins): Recognize specific antigens via a lock-and-key mechanism.
• Transport proteins: Hemoglobin (O₂ transport), Transferrin (iron transport), Albumin (general carrier in plasma).
• Storage proteins: Ferritin (iron storage in liver).
• Hormones: Insulin, glucagon, somatostatin.

Nobel Prize 1962 (Chemistry): Awarded jointly to John Kendrew (structure of myoglobin) and Max Perutz (structure of hemoglobin) “for their studies of the structures of globular proteins” — the first protein structures ever solved by X-ray crystallography.

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12. Protein Folding, Denaturation, and Proteostasis

Protein Folding

The process by which a newly synthesized polypeptide acquires its correct native 3D structure is called folding. It is essentially a self-assembly process — the information for the correct fold is entirely encoded in the primary sequence (Anfinsen’s principle).

Protein Denaturation

Denaturation is the loss of the native 3D structure — secondary, tertiary, and quaternary structures are disrupted — while the primary structure (peptide bonds) remains intact.

Causes of denaturation:

• Temperature increase (heat disrupts weak noncovalent interactions)
• Extreme pH (alters ionization state of charged groups)
• Mechanical stress
• Detergents (disrupt hydrophobic interactions)
• Chaotropic agents (e.g., urea, guanidinium chloride — disrupt hydrogen bonds and the hydrophobic effect)

The denatured protein loses its function because it loses its shape.

Anfinsen’s Experiment (Nobel Prize 1972)

Christian Anfinsen demonstrated that denaturation can be reversible:

1. Native ribonuclease A (with 4 disulfide bonds, fully active) was denatured by urea (which disrupts H-bonds and the hydrophobic effect) and reduced by β-mercaptoethanol (RSH, which breaks disulfide bonds) → completely unfolded, inactive protein with 8 free –SH groups.
2. Removal of urea and RSH (dialysis) allowed the protein to refold spontaneously → native structure and full enzymatic activity were restored.
3. Conclusion: The native structure is thermodynamically the most stable and is specified entirely by the amino acid sequence.

Proteostasis (Protein Homeostasis)

In the cell, protein folding is not always spontaneous — it can be error-prone, especially for large, complex proteins. The cell maintains protein quality control through:

1. Molecular Chaperones (Heat Shock Proteins — HSPs): Assist in correct folding and prevent aberrant aggregation. HSPs are upregulated in response to heat stress and other proteotoxic conditions.

   • Chaperones (e.g., Hsp70): Bind to exposed hydrophobic regions of unfolded proteins and prevent aggregation.
   • Chaperonins (e.g., GroEL/GroES in bacteria; Hsp60 in mitochondria): Form barrel-shaped structures that provide an isolated environment for proteins to fold correctly.

2. Endoplasmic Reticulum Associated Degradation (ERAD): Misfolded proteins in the ER are retrotranslocated to the cytoplasm and degraded.

3. Ubiquitin-Proteasome System (UPS): Misfolded or unwanted cytoplasmic proteins are tagged with ubiquitinchains and degraded by the proteasome (a large cylindrical protease complex).

4. Autophagy: Large protein aggregates and damaged organelles are engulfed by autophagosomes and delivered to lysosomes for degradation by hydrolytic enzymes.

Clinical relevance: Disruption of proteostasis underlies many diseases, including Alzheimer’s disease (amyloid plaques), Parkinson’s disease (Lewy bodies), and cystic fibrosis (misfolded CFTR protein).

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13. Post-Translational Modifications (PTMs)

After translation, many proteins are further chemically modified before they become fully functional. These post-translational modifications (PTMs) modify specific amino acid side chains and can affect:

• Protein activity
• Localization
• Stability
• Interaction with other molecules

Common Post-Translational Modifications

Modification	Description	Example
Glycosylation	Addition of carbohydrate chains	Collagen (O-linked), Immunoglobulins (N-linked)
Hydroxylation	Addition of –OH group	Pro → 4-Hydroxyproline in collagen
Phosphorylation	Addition of –PO₄ group to Ser, Thr, or Tyr	Regulation of enzyme activity
Disulfide bond formation	Oxidation of two Cys –SH groups	Insulin maturation
Proteolytic cleavage	Removal of signal peptides or propeptides	Proinsulin → Insulin

Example — Insulin: Insulin is initially synthesized as a single-chain precursor, proinsulin. Proteolytic cleavage removes the C-peptide, leaving two chains (A and B) held together by disulfide bonds — the mature, active form.

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14. Conjugated Proteins and Prosthetic Groups

Simple vs. Conjugated Proteins

• Simple proteins contain only amino acids (e.g., ribonuclease A, chymotrypsin).
• Conjugated proteins contain amino acids plus a non-amino acid component, called the prosthetic group, permanently associated with the protein.

Classification by Prosthetic Group

Class	Prosthetic Group	Example
Lipoproteins	Lipids	β₁-Lipoprotein of blood
Glycoproteins	Carbohydrates	Immunoglobulin G
Phosphoproteins	Phosphate groups	Casein of milk
Hemoproteins	Heme (iron porphyrin)	Hemoglobin, Myoglobin
Flavoproteins	Flavin nucleotides (FAD/FMN)	Succinate dehydrogenase
Metalloproteins	Iron	Ferritin
	Zinc	Alcohol dehydrogenase
	Calcium	Calmodulin
	Molybdenum	Dinitrogenase
	Copper	Plastocyanin

Note on Flavin nucleotides: FAD/FMN are stronger oxidizing agents than NAD⁺ and are particularly useful because they can participate in both one-electron and two-electron transfer reactions, making them versatile redox cofactors.

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15. Classification of Proteins

Proteins can be classified in several overlapping ways:

By Chemical Composition

• Simple: Hydrolysis yields only amino acids.
• Conjugated: Hydrolysis yields amino acids plus a prosthetic group (see Section 14).

By Shape and Solubility

• Fibrous proteins: Long, filamentous structures; insoluble in water; structural roles (e.g., collagen, keratin).
• Globular proteins: Compact, roughly spherical; usually water-soluble; functional roles (enzymes, hormones, antibodies).
• Membrane proteins: Associated with biological membranes (see Section 16).

Summary Table: Key Fibrous Proteins

Protein	Secondary Structure	Characteristics	Occurrence
α-Keratin	α-helix, crosslinked by S–S	Tough, insoluble, varying hardness	Hair, feathers, nails, horns
Silk Fibroin	β-conformation	Soft, flexible filaments	Silk
Collagen	Collagen triple helix	High tensile strength	Tendons, bone matrix

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16. Membrane Proteins

Membrane proteins are associated with the lipid bilayer and can be classified by how they associate with it:

Types of Membrane Protein Association

1. Transmembrane proteins (integral membrane proteins):

   • Span the entire lipid bilayer (single-pass or multi-pass).
   • Use hydrophobic α-helices as their transmembrane segments.
   • Can also span the membrane as a β-barrel (a rolled-up β-sheet of ~8–22 β-strands) — common in outer membrane proteins of bacteria and in mitochondria/chloroplast outer membranes.

2. Lipid-anchored proteins:

   • Attached to the membrane via a covalent lipid modification (e.g., prenylation, palmitoylation, GPI anchor) on either the cytosolic or extracellular face.
   • The protein itself does not enter the bilayer.

3. Peripheral membrane proteins:

   • Associate with the membrane non-covalently through interactions with lipid head groups or with integral membrane proteins.
   • Found on either the cytosolic or extracellular face.

4. Amphipathic α-helix proteins:

   • Anchor to the cytosolic leaflet via an amphipathic α-helix: one face of the helix is hydrophobic (inserts into the bilayer), the other is hydrophilic (faces the cytoplasm).

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17. Summary and Clinical Connections

Conceptual Summary

Structural Level	Defined by	Stabilized by
Primary	Amino acid sequence	Peptide bonds (covalent)
Secondary	Local backbone folding	Hydrogen bonds
Tertiary	Overall 3D fold	H-bonds, hydrophobic effect, van der Waals, ionic, disulfide bonds
Quaternary	Multi-subunit assembly	Same as tertiary (non-covalent, ± disulfide)

Key Principles to Remember

1. Form follows function: A protein’s 3D shape determines what it does.
2. The primary sequence contains all folding information (Anfinsen’s dogma).
3. The hydrophobic effect is the dominant driving force for folding.
4. Even a single amino acid change can destroy function (sickle cell anemia).
5. Vitamin C is essential for collagen biosynthesis (scurvy).
6. Disulfide bonds stabilize extracellular proteins; the reducing cytoplasm keeps intracellular proteins disulfide-free.
7. Denaturation disrupts secondary/tertiary/quaternary structure but not primary structure.

Clinical Connections

Disease	Protein Involved	Molecular Defect
Sickle Cell Anemia	Hemoglobin (β-chain)	Single amino acid substitution: Glu6Val
Scurvy	Collagen	Vitamin C deficiency → no Hyp/Hyl → unstable triple helix
Alzheimer’s Disease	Amyloid-β, Tau	Misfolding and aggregation (proteostasis failure)
Ehlers-Danlos Syndrome	Collagen (various types)	Mutations in collagen genes or processing enzymes
Permanent wave (application)	α-Keratin	Controlled breaking and reforming of disulfide bonds

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This lesson was prepared for General Biochemistry Module 1, Università di Bologna. All content is based on official course lecture materials.