Protein Chemistry

PPT 3

1️⃣ Page 1 – Title: Molecular Forces and Protein Folding

Image: Unfolded green chain → folded compact structure.

Meaning: Proteins go from a random coil (unfolded) to a well-defined 3D structure (native state). This transformation is governed by molecular forces and thermodynamics.

2️⃣ Page 2 – Overview of Topics

The lecture covers:

Covalent bonds
Noncovalent interactions
Electrostatics
Hydrophobicity
Folding energetics
Calorimetry
Kinetics
Folding pathways
Chaperones

This means we move from basic forces → to whole-protein thermodynamics and folding behavior.

3️⃣ Page 3 – Covalent Bonds: Potential Energy Curve

Table shows bond lengths and bond enthalpies.

Example:

C–C: 348 kJ/mol
C=C: 614 kJ/mol
C≡C: 839 kJ/mol

Graph explanation:

Y-axis: Potential energy X-axis: Interatomic distance

At large distance → energy ≈ 0 (no interaction)
As atoms approach → energy decreases (attraction)
At minimum → optimal bond length
Too close → energy rises steeply (repulsion from electron overlap)

🔑 Covalent bonds are very strong compared to noncovalent interactions.

4️⃣ Page 4 – Post-Translational Modifications

Disulfide bond formation (Cys–S–S–Cys)

Forms via oxidation of thiols
Stabilizes protein structure
Stable in neutral to acidic conditions

Other PTMs:

Glycosylation
Phosphorylation
Acylation
Proteolysis

These modify stability, activity, localization.

5️⃣ Page 5 – Charge-Charge Interactions (Coulomb's Law)

Electrostatic energy:

E ∝ (Z₁Z₂) / (D r)

D = dielectric constant

Dielectric values:

Vacuum: 1
Nonpolar solvent: 2
Water: 80

Meaning:

Water strongly weakens electrostatic interactions.

Ion-ion interaction ~60 kJ/mol in vacuum → much weaker in water.

6️⃣ Page 6 – Electrostatics on Protein Surface

Image: Myoglobin surface with ion pairs.

Charged residues form:

Salt bridges
Ion pairs

These stabilize structure, especially on surfaces.

7️⃣ Page 7 – Ionic Strength Effects

Three regimes:

Low ionic strength

Charges unshielded
Can repel → denaturation

Moderate ionic strength

Shielding stabilizes
Protein soluble

High ionic strength

Water competition
Precipitation (“salting out”)

8️⃣ Page 8 – pH and Unfolding

Low pH:

Carboxyl groups protonated
Lose negative charge

High pH:

Amino, imidazole, phenol deprotonated

Result:

Charge repulsion
Protein unfolds

Graph shows favorable vs unfavorable charge arrangements.

9️⃣ Page 9 – Charge-Dipole Interactions

Weaker than ion-ion (~8 kJ/mol).

Energy depends on:

Distance (1/r²)
Orientation (θ)

Important for:

Ion hydration
Protein–water interactions

🔟 Page 10 – Induced Dipoles & Dispersion

Temporary fluctuations in electron clouds → London dispersion forces.

All atoms experience these.

Stronger in:

Large atoms
Polarizable groups

1️⃣1️⃣ Page 11 – Summary of Electrostatics

Strength order:

Ion–ion (~60 kJ/mol) Ion–dipole Dipole–dipole Induced dipole Dispersion

Distance dependence increases rapidly:

Ion-ion ~1/r
Dispersion ~1/r⁶

1️⃣2️⃣ Page 12 – van der Waals Potential

Graph shows:

E = A/r¹² − B/r⁶

Blue curve:

Attraction at moderate distance
Repulsion at short distance

Minimum = van der Waals contact distance.

This is the balance of:

Attractive dispersion
Repulsive electron overlap

1️⃣3️⃣ Page 13 – Polarizability

Table shows:

Larger atoms → more polarizable
More outer electrons → stronger dispersion

Sulfur > oxygen > hydrogen

Important in hydrophobic cores.

1️⃣4️⃣ Page 14 – vdW Radii

Optimal H–O distance:

Sum of radii (~1.4 + 1.7 Å ≈ 3.1 Å)

Graph shows:

Too close → repulsion spike
Optimal distance → energy minimum

1️⃣5️⃣ Page 15 – Hydrogen Bonds

Energy: 4–13 kJ/mol

Distance: ~1 Å shorter than vdW contact

Directional (~180° ideal angle)

Hydrogen donor shares partial electron density with acceptor.

1️⃣6️⃣ Page 16 – Electronegativity & Donor/Acceptor Tables

Higher electronegativity:

Stronger hydrogen bonding

O > N > S

Shows common donor–acceptor pairs and typical bond lengths.

1️⃣7️⃣ Page 17 – Peptide Bond Dipole

Peptide bond:

Partial positive on N
Partial negative on O

Planarity due to resonance.

These dipoles interact in helices.

1️⃣8️⃣ Page 18 – α-Helix Macro-Dipole

Helix has:

Positive N-terminus
Negative C-terminus

Due to aligned peptide dipoles.

Explains:

Why acidic residues often near N-term
Basic residues near C-term

1️⃣9️⃣ Page 19 – Hydrogen Bonds in Stability

Interior H-bonds:

Replace water interactions
Stabilize core

Breaking internal H-bonds costs 3–5 kcal/mol.

Water competes strongly.

2️⃣0️⃣ Page 21 – Water & Hydrophobic Effect

Water hydrogen-bonds extensively.

Water weakens polar interactions.

Hydrophobic groups cluster → hydrophobic effect.

2️⃣1️⃣ Page 22 – Hydrophobicity

Buried CH₂ group: ~6.2 kJ/mol stabilization.

Oil-water analogy.

Proteins bury hydrophobic residues in core.

2️⃣2️⃣ Page 23 – Water Structure

Water network dynamic.

Hydrogen bonds constantly form/break.

2️⃣3️⃣ Page 24 – Entropic Basis of Hydrophobic Effect

Water around hydrophobic groups:

Ordered
Entropically unfavorable

When hydrophobic groups cluster:

Water released
Entropy increases
Folding favored

2️⃣4️⃣ Page 25 – ΔG = ΔH − TΔS

Graph explanation:

When hydrophobic solutes cluster:

ΔS positive (water freedom increases)
ΔG becomes negative

Folding driven by entropy of water.

2️⃣5️⃣ Page 27 – Hydrophobic Index

Ranking from most hydrophobic: Trp, Ile, Phe, Leu...

Used to predict:

Membrane helices
Transmembrane segments

2️⃣6️⃣ Page 28 – Free Energy vs Surface Area

Graphs show:

Transfer free energy correlates with accessible surface area.

Larger hydrophobic surface → larger ΔG.

2️⃣7️⃣ Page 30 – Proteins Are Dynamic

Ubiquitin NMR ensemble:

Shows multiple conformations
Termini disordered

Proteins are not rigid.

2️⃣8️⃣ Page 31 – Timescales of Motion

Range: 10⁻¹⁵ s (bond vibration) to seconds (folding)

Entropy from flexibility contributes to stability.

2️⃣9️⃣ Page 32 – Levinthal’s Paradox

Random search impossible: 100 residues → 10³⁰ conformations.

Folding must follow pathways.

3️⃣0️⃣ Page 33 – Folding Pathway Simulation

Preformed structural elements guide folding.

Energy landscape funnel model.

3️⃣1️⃣ Page 34 – Force Fields

Computational folding uses:

Bonded terms
Electrostatics
vdW
Torsion angles

3️⃣2️⃣ Page 36 – Millisecond Folding

Simulation of NTL9 for 1.5 ms.

Shows computational power.

3️⃣3️⃣ Page 37 – Chemical Denaturation

GdmCl unfolding.

Two-state: N ⇌ U

K = U/N

3️⃣4️⃣ Page 38 – Two-State Analogy

AcOH ⇌ AcO⁻ example.

Only two species present.

3️⃣5️⃣ Page 39 – Thermal Denaturation

Melting temperature (Tm): N = U

Midpoint of transition.

3️⃣6️⃣ Page 40 – Cooperativity

Steep unfolding curve.

All-or-none behavior.

3️⃣7️⃣ Page 41 – Folding Kinetics

Rate constants: kf (folding) ku (unfolding)

Equilibrium: K = kf / ku

3️⃣8️⃣ Page 42 – ΔG of Folding

ΔG = −RT lnK

Typical ΔG: −20 to −60 kJ/mol

Only equivalent to a few hydrogen bonds!

3️⃣9️⃣ Page 43 – Energy Diagram

Free energy landscape:

Unfolded → transition state → folded

ΔG‡ = activation barrier

Stability depends on ΔG.

4️⃣0️⃣ Page 44–45 – RNase Example

ΔH = −95 kcal/mol TΔS = −85 kcal/mol ΔG = −10 kcal/mol

Large enthalpy and entropy cancel → small ΔG.

Keq ≈ 2.2 × 10⁷

Protein mostly folded.

4️⃣1️⃣ Page 46 – Protein Comparisons

ΔH varies greatly. ΔG varies little.

Huge enthalpy–entropy compensation.

4️⃣2️⃣ Page 47–48 – Contributions to ΔG

Enthalpy:

Electrostatics
H-bonds
vdW
Disulfide

Entropy:

Chain entropy (unfavorable)
Hydrophobic effect (favorable)

Balance yields small net ΔG.

4️⃣3️⃣ Page 49 – Measuring Thermodynamics

ΔG from equilibrium.

ΔH & ΔS: Van’t Hoff equation.

Calorimetry measures ΔH directly.

4️⃣4️⃣ Page 50 – Differential Scanning Calorimetry

Graph shows:

Heat capacity vs temperature.

Peak area = ΔH.

Tm = peak midpoint.

4️⃣5️⃣ Page 51 – Heat Capacity Change

ΔCp increases upon unfolding.

Reason: Water orders around exposed hydrophobic surfaces.

ΔCp ~ residues × 50 J/mol·K

4️⃣6️⃣ Final Pages – Temperature Dependence

ΔG vs temperature curve:

Protein most stable at intermediate T.

At: Low T → cold denaturation High T → heat denaturation

Tm when ΔG = 0.

🧠 Overall Big Picture

Protein folding is governed by:

Strong forces:

Covalent bonds

Weak but numerous forces:

Electrostatics
Hydrogen bonds
van der Waals
Hydrophobic effect

Thermodynamics:

Large ΔH and ΔS
Small net ΔG
Cooperative transitions

Folding is:

Fast
Directed
Funnel-shaped energy landscape
Not random

Quiz

Score: 0/30 (0%)

Q0. Which interaction is strongest in vacuum according to the lecture?

Dispersion interaction

Hydrogen bond

Ion-ion interaction

Dipole-dipole interaction

Q1. How does the dielectric constant of water affect electrostatic interactions?

Strengthens them

Has no effect

Reverses their sign

Screens and weakens them

Q2. The minimum in a Lennard-Jones potential represents:

Maximum repulsion

Optimal van der Waals contact distance

Covalent bond formation

Complete electron overlap

Q3. Typical hydrogen bond strength in proteins is:

0.1–1 kJ/mol

4–13 kJ/mol

50–100 kJ/mol

200–400 kJ/mol

Q4. The α-helix macro-dipole results from:

Side chain ionization

Aligned peptide bond dipoles

Disulfide bonds

Water ordering

Q5. The hydrophobic effect is primarily driven by:

Enthalpy gain of side chains

Entropy increase of water

Covalent bond formation

Salt bridge formation

Q6. Levinthal’s paradox demonstrates that protein folding:

Is random

Is thermodynamically impossible

Cannot occur by random conformational search

Requires covalent bond rearrangement

Q7. At the melting temperature (Tm):

Protein is fully unfolded

Protein is fully folded

[Folded] = [Unfolded]

ΔH = 0

Q8. Typical ΔG° of folding for small globular proteins is:

−5 to −14 kJ/mol

−20 to −60 kJ/mol

−200 to −500 kJ/mol

0 kJ/mol

Q9. In a two-state unfolding model:

Multiple intermediates dominate

Only folded and unfolded states are populated

The transition is irreversible

ΔG is zero at all temperatures

Q10. The van’t Hoff equation allows determination of:

ΔG only

ΔH and ΔS from temperature dependence of Keq

Bond lengths

Heat capacity directly

Q11. Heat capacity (Cp) increases upon unfolding mainly because:

Covalent bonds break

Water becomes more ordered around exposed hydrophobic surfaces

Protein mass increases

Ionic strength changes

Q12. Burial of a methylene group contributes approximately:

~0.5 kJ/mol

~6 kJ/mol

~60 kJ/mol

~600 kJ/mol

Q13. The folding equilibrium constant is related to rate constants as:

K = ku/kf

K = kf + ku

K = kf/ku

K = 1/(kf·ku)

Q14. Dispersion interactions arise from:

Permanent dipoles

Covalent bonding

Correlated electron density fluctuations

Hydrogen transfer

Q15. Water weakens hydrogen bonds between polar groups in proteins.

True

False

Q16. Hydrophobic side chains can directly hydrogen bond with water.

True

False

Q17. ΔG° = −RT ln Keq.

True

False

Q18. Protein folding is driven purely by enthalpy.

True

False

Q19. Disulfide bonds contribute covalent stabilization to protein structure.

True

False

Q20. At very high ionic strength, proteins may precipitate due to reduced solvation.

True

False

Q21. Hydrogen bonds are typically longer than van der Waals contact distances.

True

False

Q22. The unfolded state always has lower entropy than the folded state.

True

False

Q23. Levinthal’s paradox implies folding requires specific pathways.

True

False

Q24. Electrostatic interactions scale as 1/r in ion-ion interactions.

True

False

Q25. At Tm, ΔG of unfolding equals zero.

True

False

Q26. Heat capacity changes upon unfolding are negligible.

True

False

Q27. Hydrophobic index scales can be used to predict membrane helices.

True

False

Q28. Enthalpy–entropy compensation is common in protein folding.

True

False

Q29. Folding is typically a non-cooperative gradual process over a wide temperature range.

True

False