Four Levels of Protein Structure:

1. Primary Structure

(a) Bonds: Peptide bonds (covalent) linking amino acids

• Sequence of amino acids in polypeptide chain
• Written N-terminus to C-terminus
• Determined by DNA sequence (gene)

(b) Example: Insulin A-chain: 21 amino acids starting with Gly-Ile-Val-Glu...

2. Secondary Structure

(a) Bonds/Interactions: Hydrogen bonds between backbone atoms (C=O and N-H)

Two main types:

• α-helix: Coiled structure, H-bonds between every 4th amino acid
• β-pleated sheet: Extended strands, H-bonds between adjacent strands (parallel or antiparallel)

(b) Example:

• α-helix: Keratin in hair, myoglobin
• β-sheet: Silk fibroin (antiparallel)

3. Tertiary Structure

(a) Bonds/Interactions:

• Hydrophobic interactions: Nonpolar R-groups cluster in interior
• Hydrogen bonds: Between R-groups
• Ionic bonds (salt bridges): Between charged R-groups (+ and -)
• Disulfide bridges (S-S): Covalent bonds between cysteine residues
• van der Waals forces: Weak attractions

Overall 3D shape of single polypeptide

(b) Example:

• Lysozyme (enzyme): specific 3D shape creates active site
• Myoglobin: globular protein with heme group

4. Quaternary Structure

(a) Bonds/Interactions: Same as tertiary (H-bonds, ionic, hydrophobic, van der Waals)

• Multiple polypeptide subunits associate
• Not all proteins have quaternary structure
• Functional protein complex

(b) Example:

• Hemoglobin: 4 subunits (2α, 2β chains), each with heme
• Collagen: 3 polypeptide helices twisted together (triple helix)

Summary Table:

| Level | Bond/Interaction | Example | |-------|-----------------|---------| | 1° | Peptide bonds | Amino acid sequence | | 2° | H-bonds (backbone) | α-helix, β-sheet | | 3° | Multiple (R-groups) | Myoglobin (3D fold) | | 4° | Multiple (subunits) | Hemoglobin (4 subunits) |

$\boxed{\text{1°: sequence, 2°: local folds, 3°: 3D shape, 4°: multiple chains}}$

Key Concept: Structure determines function! Denaturation (loss of 3D structure) → loss of function.

Four Levels of Protein Structure:

1. Primary Structure

(a) Bonds: Peptide bonds (covalent) linking amino acids

• Sequence of amino acids in polypeptide chain
• Written N-terminus to C-terminus
• Determined by DNA sequence (gene)

(b) Example: Insulin A-chain: 21 amino acids starting with Gly-Ile-Val-Glu...

2. Secondary Structure

(a) Bonds/Interactions: Hydrogen bonds between backbone atoms (C=O and N-H)

Two main types:

• α-helix: Coiled structure, H-bonds between every 4th amino acid
• β-pleated sheet: Extended strands, H-bonds between adjacent strands (parallel or antiparallel)

(b) Example:

• α-helix: Keratin in hair, myoglobin
• β-sheet: Silk fibroin (antiparallel)

3. Tertiary Structure

(a) Bonds/Interactions:

• Hydrophobic interactions: Nonpolar R-groups cluster in interior
• Hydrogen bonds: Between R-groups
• Ionic bonds (salt bridges): Between charged R-groups (+ and -)
• Disulfide bridges (S-S): Covalent bonds between cysteine residues
• van der Waals forces: Weak attractions

Overall 3D shape of single polypeptide

(b) Example:

• Lysozyme (enzyme): specific 3D shape creates active site
• Myoglobin: globular protein with heme group

4. Quaternary Structure

(a) Bonds/Interactions: Same as tertiary (H-bonds, ionic, hydrophobic, van der Waals)

• Multiple polypeptide subunits associate
• Not all proteins have quaternary structure
• Functional protein complex

(b) Example:

• Hemoglobin: 4 subunits (2α, 2β chains), each with heme
• Collagen: 3 polypeptide helices twisted together (triple helix)

Summary Table:

| Level | Bond/Interaction | Example | |-------|-----------------|---------| | 1° | Peptide bonds | Amino acid sequence | | 2° | H-bonds (backbone) | α-helix, β-sheet | | 3° | Multiple (R-groups) | Myoglobin (3D fold) | | 4° | Multiple (subunits) | Hemoglobin (4 subunits) |

$\boxed{\text{1°: sequence, 2°: local folds, 3°: 3D shape, 4°: multiple chains}}$

Key Concept: Structure determines function! Denaturation (loss of 3D structure) → loss of function.

Four Levels of Protein Structure:

1. Primary Structure

(a) Bonds: Peptide bonds (covalent) linking amino acids

• Sequence of amino acids in polypeptide chain
• Written N-terminus to C-terminus
• Determined by DNA sequence (gene)

(b) Example: Insulin A-chain: 21 amino acids starting with Gly-Ile-Val-Glu...

2. Secondary Structure

(a) Bonds/Interactions: Hydrogen bonds between backbone atoms (C=O and N-H)

Two main types:

• α-helix: Coiled structure, H-bonds between every 4th amino acid
• β-pleated sheet: Extended strands, H-bonds between adjacent strands (parallel or antiparallel)

(b) Example:

• α-helix: Keratin in hair, myoglobin
• β-sheet: Silk fibroin (antiparallel)

3. Tertiary Structure

(a) Bonds/Interactions:

• Hydrophobic interactions: Nonpolar R-groups cluster in interior
• Hydrogen bonds: Between R-groups
• Ionic bonds (salt bridges): Between charged R-groups (+ and -)
• Disulfide bridges (S-S): Covalent bonds between cysteine residues
• van der Waals forces: Weak attractions

Overall 3D shape of single polypeptide

(b) Example:

• Lysozyme (enzyme): specific 3D shape creates active site
• Myoglobin: globular protein with heme group

4. Quaternary Structure

(a) Bonds/Interactions: Same as tertiary (H-bonds, ionic, hydrophobic, van der Waals)

• Multiple polypeptide subunits associate
• Not all proteins have quaternary structure
• Functional protein complex

(b) Example:

• Hemoglobin: 4 subunits (2α, 2β chains), each with heme
• Collagen: 3 polypeptide helices twisted together (triple helix)

Summary Table:

| Level | Bond/Interaction | Example | |-------|-----------------|---------| | 1° | Peptide bonds | Amino acid sequence | | 2° | H-bonds (backbone) | α-helix, β-sheet | | 3° | Multiple (R-groups) | Myoglobin (3D fold) | | 4° | Multiple (subunits) | Hemoglobin (4 subunits) |

$\boxed{\text{1°: sequence, 2°: local folds, 3°: 3D shape, 4°: multiple chains}}$

Key Concept: Structure determines function! Denaturation (loss of 3D structure) → loss of function.

Four Levels of Protein Structure:

1. Primary Structure

(a) Bonds: Peptide bonds (covalent) linking amino acids

• Sequence of amino acids in polypeptide chain
• Written N-terminus to C-terminus
• Determined by DNA sequence (gene)

(b) Example: Insulin A-chain: 21 amino acids starting with Gly-Ile-Val-Glu...

2. Secondary Structure

(a) Bonds/Interactions: Hydrogen bonds between backbone atoms (C=O and N-H)

Two main types:

• α-helix: Coiled structure, H-bonds between every 4th amino acid
• β-pleated sheet: Extended strands, H-bonds between adjacent strands (parallel or antiparallel)

(b) Example:

• α-helix: Keratin in hair, myoglobin
• β-sheet: Silk fibroin (antiparallel)

3. Tertiary Structure

(a) Bonds/Interactions:

• Hydrophobic interactions: Nonpolar R-groups cluster in interior
• Hydrogen bonds: Between R-groups
• Ionic bonds (salt bridges): Between charged R-groups (+ and -)
• Disulfide bridges (S-S): Covalent bonds between cysteine residues
• van der Waals forces: Weak attractions

Overall 3D shape of single polypeptide

(b) Example:

• Lysozyme (enzyme): specific 3D shape creates active site
• Myoglobin: globular protein with heme group

4. Quaternary Structure

(a) Bonds/Interactions: Same as tertiary (H-bonds, ionic, hydrophobic, van der Waals)

• Multiple polypeptide subunits associate
• Not all proteins have quaternary structure
• Functional protein complex

(b) Example:

• Hemoglobin: 4 subunits (2α, 2β chains), each with heme
• Collagen: 3 polypeptide helices twisted together (triple helix)

Summary Table:

| Level | Bond/Interaction | Example | |-------|-----------------|---------| | 1° | Peptide bonds | Amino acid sequence | | 2° | H-bonds (backbone) | α-helix, β-sheet | | 3° | Multiple (R-groups) | Myoglobin (3D fold) | | 4° | Multiple (subunits) | Hemoglobin (4 subunits) |

$\boxed{\text{1°: sequence, 2°: local folds, 3°: 3D shape, 4°: multiple chains}}$

Key Concept: Structure determines function! Denaturation (loss of 3D structure) → loss of function.

Enzyme Conditions: Optimal at pH 7.0 and 37°C

(a) pH changed to 3.0 (strongly acidic):

Prediction: ⚠️ Enzyme activity greatly reduced or eliminated

Explanation:

1. Protonation of amino acids:

   • Acidic pH adds excess H⁺ ions
   • Amino acid R-groups become protonated
   • Charged residues (Asp⁻, Glu⁻) become neutral (AspH, GluH)
   • Basic residues (His, Lys, Arg) become more positive

2. Disruption of ionic bonds:

   • Salt bridges (electrostatic interactions) break
   • Changes in charge distribution

3. Tertiary structure denaturation:

   • 3D shape distorts
   • Active site changes shape
   • Substrate can no longer bind properly

4. Result: Loss of catalytic activity (may be reversible if pH restored quickly)

(b) Temperature increased to 80°C (far above optimum):

Prediction: ⚠️ Enzyme denatured, activity lost permanently

Explanation:

1. Increased kinetic energy:

   • Molecules vibrate more vigorously
   • Weak bonds break (H-bonds, ionic, hydrophobic)

2. Progressive unfolding:

   • Secondary structure disrupted (α-helices, β-sheets unfold)
   • Tertiary structure lost
   • Protein unfolds into random coil

3. Permanent denaturation:

   • Polypeptide chains may aggregate
   • Disulfide bonds may scramble
   • Irreversible damage

4. Activity-Temperature Relationship:

   Activity
     ^
     |     /\
     |    /  \
     |   /    \_____ (denaturation)
     |  /
     |_/________________> Temperature
         37°C  80°C
   

Why irreversible: Unlike pH change, heat breaks so many bonds simultaneously that protein cannot refold to native state.

(c) Competitive inhibitor added:

Prediction: 🔽 Enzyme activity reduced but NOT eliminated

Explanation:

1. Competitive inhibition mechanism:

   • Inhibitor structurally similar to substrate
   • Competes for same active site
   • Reversibly binds to enzyme

2. Effect on protein structure:

   • No structural change to enzyme!
   • Enzyme remains properly folded
   • Active site unchanged

3. Kinetic effects:

   • ↑ K_m (apparent affinity for substrate decreases)
   • V_max unchanged (can be overcome with excess substrate)

4. Equation:

   $v = \frac{V_{max}[S]}{K_m(1 + [I]/K_i) + [S]}$

   where [I] = inhibitor concentration, K_i = inhibitor constant

5. Key difference:

   • Can be overcome by increasing substrate concentration
   • At high [S], substrate outcompetes inhibitor
   • Eventually reaches V_max

Comparison:

| Condition | Structure Change | Activity | Reversible? | |-----------|-----------------|----------|-------------| | Low pH | Tertiary disrupted | Very low | Yes (if quick) | | High temp | Complete denaturation | Zero | No | | Competitive inh. | None | Reduced | Yes (↑ substrate) |

$\boxed{\text{(a) Denatures (reversible), (b) Denatures (irreversible), (c) Active site blocked (reversible)}}$

Enzyme Conditions: Optimal at pH 7.0 and 37°C

(a) pH changed to 3.0 (strongly acidic):

Prediction: ⚠️ Enzyme activity greatly reduced or eliminated

Explanation:

1. Protonation of amino acids:

   • Acidic pH adds excess H⁺ ions
   • Amino acid R-groups become protonated
   • Charged residues (Asp⁻, Glu⁻) become neutral (AspH, GluH)
   • Basic residues (His, Lys, Arg) become more positive

2. Disruption of ionic bonds:

   • Salt bridges (electrostatic interactions) break
   • Changes in charge distribution

3. Tertiary structure denaturation:

   • 3D shape distorts
   • Active site changes shape
   • Substrate can no longer bind properly

4. Result: Loss of catalytic activity (may be reversible if pH restored quickly)

(b) Temperature increased to 80°C (far above optimum):

Prediction: ⚠️ Enzyme denatured, activity lost permanently

Explanation:

1. Increased kinetic energy:

   • Molecules vibrate more vigorously
   • Weak bonds break (H-bonds, ionic, hydrophobic)

2. Progressive unfolding:

   • Secondary structure disrupted (α-helices, β-sheets unfold)
   • Tertiary structure lost
   • Protein unfolds into random coil

3. Permanent denaturation:

   • Polypeptide chains may aggregate
   • Disulfide bonds may scramble
   • Irreversible damage

4. Activity-Temperature Relationship:

   Activity
     ^
     |     /\
     |    /  \
     |   /    \_____ (denaturation)
     |  /
     |_/________________> Temperature
         37°C  80°C
   

Why irreversible: Unlike pH change, heat breaks so many bonds simultaneously that protein cannot refold to native state.

(c) Competitive inhibitor added:

Prediction: 🔽 Enzyme activity reduced but NOT eliminated

Explanation:

1. Competitive inhibition mechanism:

   • Inhibitor structurally similar to substrate
   • Competes for same active site
   • Reversibly binds to enzyme

2. Effect on protein structure:

   • No structural change to enzyme!
   • Enzyme remains properly folded
   • Active site unchanged

3. Kinetic effects:

   • ↑ K_m (apparent affinity for substrate decreases)
   • V_max unchanged (can be overcome with excess substrate)

4. Equation:

   $v = \frac{V_{max}[S]}{K_m(1 + [I]/K_i) + [S]}$

   where [I] = inhibitor concentration, K_i = inhibitor constant

5. Key difference:

   • Can be overcome by increasing substrate concentration
   • At high [S], substrate outcompetes inhibitor
   • Eventually reaches V_max

Comparison:

| Condition | Structure Change | Activity | Reversible? | |-----------|-----------------|----------|-------------| | Low pH | Tertiary disrupted | Very low | Yes (if quick) | | High temp | Complete denaturation | Zero | No | | Competitive inh. | None | Reduced | Yes (↑ substrate) |

$\boxed{\text{(a) Denatures (reversible), (b) Denatures (irreversible), (c) Active site blocked (reversible)}}$

Enzyme Conditions: Optimal at pH 7.0 and 37°C

(a) pH changed to 3.0 (strongly acidic):

Prediction: ⚠️ Enzyme activity greatly reduced or eliminated

Explanation:

1. Protonation of amino acids:

   • Acidic pH adds excess H⁺ ions
   • Amino acid R-groups become protonated
   • Charged residues (Asp⁻, Glu⁻) become neutral (AspH, GluH)
   • Basic residues (His, Lys, Arg) become more positive

2. Disruption of ionic bonds:

   • Salt bridges (electrostatic interactions) break
   • Changes in charge distribution

3. Tertiary structure denaturation:

   • 3D shape distorts
   • Active site changes shape
   • Substrate can no longer bind properly

4. Result: Loss of catalytic activity (may be reversible if pH restored quickly)

(b) Temperature increased to 80°C (far above optimum):

Prediction: ⚠️ Enzyme denatured, activity lost permanently

Explanation:

1. Increased kinetic energy:

   • Molecules vibrate more vigorously
   • Weak bonds break (H-bonds, ionic, hydrophobic)

2. Progressive unfolding:

   • Secondary structure disrupted (α-helices, β-sheets unfold)
   • Tertiary structure lost
   • Protein unfolds into random coil

3. Permanent denaturation:

   • Polypeptide chains may aggregate
   • Disulfide bonds may scramble
   • Irreversible damage

4. Activity-Temperature Relationship:

   Activity
     ^
     |     /\
     |    /  \
     |   /    \_____ (denaturation)
     |  /
     |_/________________> Temperature
         37°C  80°C
   

Why irreversible: Unlike pH change, heat breaks so many bonds simultaneously that protein cannot refold to native state.

(c) Competitive inhibitor added:

Prediction: 🔽 Enzyme activity reduced but NOT eliminated

Explanation:

1. Competitive inhibition mechanism:

   • Inhibitor structurally similar to substrate
   • Competes for same active site
   • Reversibly binds to enzyme

2. Effect on protein structure:

   • No structural change to enzyme!
   • Enzyme remains properly folded
   • Active site unchanged

3. Kinetic effects:

   • ↑ K_m (apparent affinity for substrate decreases)
   • V_max unchanged (can be overcome with excess substrate)

4. Equation:

   $v = \frac{V_{max}[S]}{K_m(1 + [I]/K_i) + [S]}$

   where [I] = inhibitor concentration, K_i = inhibitor constant

5. Key difference:

   • Can be overcome by increasing substrate concentration
   • At high [S], substrate outcompetes inhibitor
   • Eventually reaches V_max

Comparison:

| Condition | Structure Change | Activity | Reversible? | |-----------|-----------------|----------|-------------| | Low pH | Tertiary disrupted | Very low | Yes (if quick) | | High temp | Complete denaturation | Zero | No | | Competitive inh. | None | Reduced | Yes (↑ substrate) |

$\boxed{\text{(a) Denatures (reversible), (b) Denatures (irreversible), (c) Active site blocked (reversible)}}$

Enzyme Conditions: Optimal at pH 7.0 and 37°C

(a) pH changed to 3.0 (strongly acidic):

Prediction: ⚠️ Enzyme activity greatly reduced or eliminated

Explanation:

1. Protonation of amino acids:

   • Acidic pH adds excess H⁺ ions
   • Amino acid R-groups become protonated
   • Charged residues (Asp⁻, Glu⁻) become neutral (AspH, GluH)
   • Basic residues (His, Lys, Arg) become more positive

2. Disruption of ionic bonds:

   • Salt bridges (electrostatic interactions) break
   • Changes in charge distribution

3. Tertiary structure denaturation:

   • 3D shape distorts
   • Active site changes shape
   • Substrate can no longer bind properly

4. Result: Loss of catalytic activity (may be reversible if pH restored quickly)

(b) Temperature increased to 80°C (far above optimum):

Prediction: ⚠️ Enzyme denatured, activity lost permanently

Explanation:

1. Increased kinetic energy:

   • Molecules vibrate more vigorously
   • Weak bonds break (H-bonds, ionic, hydrophobic)

2. Progressive unfolding:

   • Secondary structure disrupted (α-helices, β-sheets unfold)
   • Tertiary structure lost
   • Protein unfolds into random coil

3. Permanent denaturation:

   • Polypeptide chains may aggregate
   • Disulfide bonds may scramble
   • Irreversible damage

4. Activity-Temperature Relationship:

   Activity
     ^
     |     /\
     |    /  \
     |   /    \_____ (denaturation)
     |  /
     |_/________________> Temperature
         37°C  80°C
   

Why irreversible: Unlike pH change, heat breaks so many bonds simultaneously that protein cannot refold to native state.

(c) Competitive inhibitor added:

Prediction: 🔽 Enzyme activity reduced but NOT eliminated

Explanation:

1. Competitive inhibition mechanism:

   • Inhibitor structurally similar to substrate
   • Competes for same active site
   • Reversibly binds to enzyme

2. Effect on protein structure:

   • No structural change to enzyme!
   • Enzyme remains properly folded
   • Active site unchanged

3. Kinetic effects:

   • ↑ K_m (apparent affinity for substrate decreases)
   • V_max unchanged (can be overcome with excess substrate)

4. Equation:

   $v = \frac{V_{max}[S]}{K_m(1 + [I]/K_i) + [S]}$

   where [I] = inhibitor concentration, K_i = inhibitor constant

5. Key difference:

   • Can be overcome by increasing substrate concentration
   • At high [S], substrate outcompetes inhibitor
   • Eventually reaches V_max

Comparison:

| Condition | Structure Change | Activity | Reversible? | |-----------|-----------------|----------|-------------| | Low pH | Tertiary disrupted | Very low | Yes (if quick) | | High temp | Complete denaturation | Zero | No | | Competitive inh. | None | Reduced | Yes (↑ substrate) |

$\boxed{\text{(a) Denatures (reversible), (b) Denatures (irreversible), (c) Active site blocked (reversible)}}$