🧬 1. Amino Acids – Core Foundations

🔹 Structure & Charges (Zwitterions)

• Free amino acids exist as zwitterions:
  • NH₃⁺ (positive) at the N-terminus
  • COO⁻ (negative) at the C-terminus
• Net charge depends on pH vs pKa

👉 Key idea: At physiological pH, amino acids are not neutral—they carry internal charges.

🔹 Chirality

• All amino acids are chiral except glycine
• Chirality → gives signal in circular dichroism (CD)

👉 Meaning: Chirality allows proteins to adopt specific 3D structures and be detected spectroscopically.

🔹 Classification

You must know groups and properties:

• Basic: Lysine, Arginine, Histidine
• Acidic: Aspartate, Glutamate
• Hydrophobic: e.g. Leucine, Valine
• Hydrophilic: e.g. Serine

👉 Important: These properties determine protein folding and function.

🔹 pKa, pH, and pI

• pKa = pH where group is 50% protonated
• pI = pH where net charge = 0

Key examples:

• Histidine (pKa ~6.4) → partially charged near physiological pH
• Cysteine (pKa ~8.3) → can become negatively charged at physiological pH

👉 Insight: Charge is not binary at population level—it's fractional across molecules.

🔹 Special Amino Acids

• Not just 20:
  • Selenocysteine
  • Pyrrolysine
• Non-natural amino acids can be synthetically introduced

👉 Importance: Expands protein function beyond natural biology.

🔹 Structure Preferences

• Proline = helix breaker
  • Rigid ring restricts backbone → disrupts α-helix
• Amino acid distribution differs in:
  • α-helices
  • β-sheets

👉 You should understand why, not just memorize.

🧱 2. Protein Structure Levels

• Primary: sequence
• Secondary: α-helix, β-sheet
• Tertiary: 3D fold
• Quaternary: multiple subunits

🔹 Hydrogen Bonding

• Stabilizes secondary structure:
  • α-helix: intra-chain H-bonds
  • β-sheet: inter-strand H-bonds

👉 Critical for exam: identify donor/acceptor roles

⚗️ 3. Chemical Reactivity & PTMs

🔹 Reactive Amino Acids

• Some are inert, others highly reactive
• Important for post-translational modifications (PTMs)

🔹 Post-Translational Modifications

1. Enzymatic:

• Phosphorylation (Ser, Thr, Tyr)
  • Adds negative charge
  • Changes structure + interactions

2. Non-enzymatic:

• Glycation (random sugar attachment)

3. Enzymatic glycosylation:

• Controlled sugar addition

👉 Key concept: PTMs change charge, polarity, and H-bonding capacity → altering protein behavior

🧪 4. Protein Sequence Determination

• Edman degradation (old method)
• Mass spectrometry (MS) (modern standard)

👉 Important:

• Know both, but MS is most relevant today
• Edman may still appear as a conceptual question

⚖️ 5. Protein Size vs Molecular Weight

🔹 Molecular Weight

• Sum of amino acids (minus water from peptide bonds)

🔹 Size (Hydrodynamic)

• Depends on shape:
  • Compact → smaller
  • Elongated → larger

👉 Key insight: Same MW ≠ same size

🔹 Methods

SDS-PAGE

• Estimates molecular weight
• Assumes uniform SDS binding

⚠️ Problem:

• Uneven binding (e.g. acidic proteins) → wrong size

Gel Filtration (Size Exclusion)

• Measures hydrodynamic size
• Done under native conditions

Mass Spectrometry

• Most accurate MW measurement

🔥 6. Protein Folding

🔹 Forces Involved

• Covalent: strong (e.g. disulfide bonds)
• Non-covalent:
  • Hydrogen bonds
  • Ionic interactions
  • van der Waals
  • Hydrophobic effect

👉 Many weak interactions → strong overall stability

🔹 Folding Principles

• Driven by energy minimization
• Forms via intermediate states

🔹 Energetics

• Folding involves:
  • Enthalpy (ΔH)
  • Entropy (ΔS)

🔹 Experimental Techniques

Differential Scanning Calorimetry (DSC)

• Measures melting temperature (Tm)
• Gives folding energetics

Denaturants

• e.g. urea
• Stabilize unfolded state

Osmolytes

• Stabilize folded state

🔹 Folding Kinetics

• Measured using stop-flow experiments
• Produces Chevron plots

👉 Important:

• Folding rate ≠ folding mechanism details

🔹 Mutational Analysis

• Identify key residues in folding
• Reveals:
  • Folding nucleus
  • Transition states

🔗 7. Ligand Binding

🔹 Binding Strength

• Kd (dissociation constant)
• Lower Kd → stronger binding

🔹 Thermodynamics

• Measured via:
  • van’t Hoff analysis
  • ITC

🔹 Cooperativity

• Binding sites can be:
  • Independent
  • Cooperative

🔹 Microscopic vs Macroscopic Kd

• Microscopic: individual sites (hard to measure)
• Macroscopic: overall binding

📊 8. Binding Measurement Techniques

🔹 Equilibrium Dialysis

• Measures free vs bound ligand

🔹 ITC (Isothermal Titration Calorimetry)

• Measures heat change
• Gives:
  • Kd
  • ΔH
  • stoichiometry

🔹 SPR (Surface Plasmon Resonance)

• Measures binding in real time
• Outputs:
  • on-rate (kon)
  • off-rate (koff)

👉 You must interpret curves, not calculate exact values

🔹 BLI (BioLayer Interferometry)

• Similar output to SPR

🔹 Thermophoresis

• Movement depends on binding state

🧼 9. Protein Purification

🔹 Pre-considerations

• Where is protein?
  • Cytoplasm
  • Periplasm
  • Inclusion bodies

🔹 Methods

Ammonium Sulfate Precipitation

• Salt competes for water → proteins aggregate

Affinity Chromatography

• Based on specific binding
• Example: His-tag

Ion Exchange Chromatography

• Separates by charge

Hydrophobic Interaction Chromatography

• High salt → exposes hydrophobic patches

Gel Filtration

• Separates by size

🧪 10. Molecular Evolution

🔹 Mutation Types

• Positive
• Neutral
• Negative

🔹 Library Generation

• Error-prone PCR
• Cassette mutagenesis
• DNA shuffling

👉 Important: know pros/cons and when to use each

🔹 Selection Pressure

• Drives evolution toward desired traits

🔹 Folding vs Function

• Mutation may:
  • Not affect activity
  • But disrupt folding

👉 Critical insight for design

🧫 11. Display & Selection Techniques

• Phage display
• Yeast display
• Ribosome display
• Compartmentalization

👉 All link: Genotype ↔ Phenotype

🧠 12. AlphaFold (Exam Focus)

You do NOT need deep details—focus on interpretation:

🔹 pLDDT

• Measures local confidence
• High = reliable structure locally

🔹 PAE (Predicted Alignment Error)

• Measures global domain placement
• Low = domains correctly positioned

🔹 Key distinction:

• pLDDT → local accuracy
• PAE → global arrangement

⚡ Final Exam Strategy Insights

• Questions often based on real papers
• Focus on:
  • Understanding principles
  • Interpreting data (graphs, spectra)
• Less emphasis on:
  • Memorizing obscure details

🧩 Key Takeaways

• Protein chemistry is about structure ↔ function ↔ energetics
• Weak forces collectively drive folding
• Modifications and mutations reshape protein behavior
• Experimental methods measure different aspects, not the same thing

🧬 1. Carboxyl group, pI, and charge

❌ Misconception:

• “Carboxyl will be positive”

✅ Correct:

• Carboxyl group (–COOH):
  • Protonated (COOH) → neutral
  • Deprotonated (COO⁻) → negative

👉 It is never positive

🔹 pI (isoelectric point)

• pH where net charge = 0
• In proteins:
  • Only side chains + termini contribute

👉 Meaning:

• Side chains can be:
  • Charged (Asp⁻, Lys⁺)
  • Uncharged (Ser, Leu)

🔗 2. Hydrogen bonds in protein structure

🔹 Donor vs Acceptor

• Donor: has H → e.g. N–H
• Acceptor: lone pair → e.g. C=O oxygen

🔹 α-helix

• H-bond:
  • C=O (residue i) → N–H (residue i+4)

🔹 β-sheet

• H-bonds between strands:
  • backbone C=O ↔ N–H

👉 Backbone, not side chains, stabilize these structures

⚗️ 3. PTMs (Post-Translational Modifications)

🔹 “Not catalyzed” means:

• Occur without enzymes

Example:

• Glycation → random sugar attachment

🔹 Enzymatic PTMs

• Glycosylation → enzyme-controlled sugar addition

🔹 Effect on amino acids

PTMs can:

• Change charge
• Change polarity
• Change size
• Change H-bonding capacity

⚡ 4. Serine phosphorylation

❌ Misconception:

• “3 hydrogens”

✅ Correct:

• Serine → OH group (neutral)
• After phosphorylation:
  • becomes –PO₃²⁻
  • carries ~2 negative charges

👉 Effects:

• Strong electrostatic changes
• More oxygen atoms → more H-bond acceptors, not hydrogens

🧪 5. Sequence determination without MS

Use:

• Edman degradation

👉 Stepwise removal of N-terminal amino acids 👉 Older, slower, but still valid conceptually

⚖️ 6. SDS-PAGE accuracy

Key idea:

• SDS binds ~uniformly → gives negative charge

❗ Problem:

• Binding is not perfect
  • acidic regions repel SDS

👉 Result:

• Migration ≠ exact molecular weight
• It is an approximation, not exact

🔹 Important correction:

• SDS-PAGE is denatured, so:
  • Shape influence is reduced
  • BUT inaccuracies still exist

🔥 7. Forces in protein folding

Most important:

• Hydrophobic effect (dominant)
• Hydrogen bonds
• Ionic interactions
• van der Waals

👉 Hydrophobic collapse drives folding core formation

🧠 8. Folding pathways

• Proteins fold toward:
  • lowest free energy (ΔG minimum)

👉 Not a single path:

• Multiple intermediates exist

🔗 9. Ligand binding and folding

• Ligand binding:
  • Stabilizes folded state
  • Makes unfolding harder

👉 Shifts equilibrium:

• Folded ↔ unfolded → favors folded

⏱️ 10. Stop-flow & Chevron plots

Process:

1. Protein in high urea (unfolded)
2. Rapid dilution
3. Measure folding rate

Chevron plot:

• X-axis: denaturant concentration
• Y-axis: log(rate)

👉 Gives:

• Folding rate (k_f)
• Unfolding rate (k_u)

🧬 11. Mutational analysis

• Mutations reveal:
  • Effects on transition state
  • Effects on folded state stability

👉 Used to locate:

• Folding nucleus

🔗 12. Two ligands binding

You get:

• Kd (dissociation constant)

🔹 Micro vs Macro

• Microscopic Kd:
  • Each binding site separately
  • Measured with NMR
• Macroscopic Kd:
  • Overall binding
  • Most experiments measure this

🧪 13. Equilibrium dialysis (ligand passes membrane)

• Membrane:
  • Ligand passes
  • Protein does NOT

👉 Measure:

• Free ligand concentration

👉 Then calculate:

• Kd

🧫 14. Gel filtration + ligand binding

Concept:

• Protein + ligand mixture

What happens:

• Protein–ligand complex:
  • Larger → elutes first
• Free ligand:
  • Smaller → elutes later

👉 You measure both → calculate binding

📡 15. SPR (Surface Plasmon Resonance)

Principle:

• Measures refractive index change at surface

Sensorgram:

• Association phase → signal increases
• Dissociation phase → signal decreases

Interpretation:

• Steep increase → fast binding
• Slow decay → strong binding

⏱️ 16. On-rate and Off-rate

• k_on = how fast ligand binds
• k_off = how fast it dissociates

👉 Relationship:

• Strong binding = high k_on + low k_off

🌈 17. BLI (BioLayer Interferometry)

Completed idea:

• Uses light interference
• Light reflects from two surfaces:
  • internal reference
  • binding layer

👉 Binding changes thickness → changes interference pattern

🧂 18. Ammonium sulfate precipitation (AMS)

Correct interpretation:

• Salt competes for water
• Water leaves protein surface

Result:

• Hydrophobic patches exposed
• Proteins aggregate → precipitate

👉 Yes, strongly linked to hydrophobicity

🧲 19. Affinity chromatography

Principle:

• Specific interaction:
  • ligand ↔ protein

Works for:

• Natural proteins
• Engineered tags (His-tag)

⚡ 20. Ion exchange chromatography

• Based on charge interactions

👉 Yes:

• It is a form of affinity (electrostatic)

💧 21. Hydrophobic interaction chromatography

Similar to AMS:

• Add salt → expose hydrophobic patches

Binding:

• Protein binds hydrophobic column

Elution:

• Decrease salt → water returns → protein released

🩸 22. Blood coagulation cascade

Key concept:

• Proteolytic activation

👉 Enzymes activate other enzymes → cascade

💊 23. Therapeutic proteins

• Designed for:
  • stability
  • specificity
  • low immunogenicity

🧬 24. Antibody mutation

• Improve:
  • affinity
  • stability
  • specificity

🔥 25. Stability vs folding problem

Key insight:

• Mutation may:
  • Not affect function
  • BUT disrupt folding

👉 Folding is prerequisite for function

🧪 26. Selection strategies (core principles)

🔹 Phage display

• Protein displayed on virus
• DNA inside → links genotype–phenotype

🔹 Yeast display

• Protein on yeast surface
• Screen via flow cytometry (FACS)

🔹 Ribosome display

• Protein + mRNA + ribosome complex
• No cell needed

🔹 Compartmentalization

• Each gene isolated in droplets
• Selection happens individually

🔹 Selection types

Affinity

• Select strongest binders

Stability

• Select most stable proteins

Activity

• Select best enzymatic function

🧠 Final Core Insight

Most of your confusion points reduce to:

• Charge ↔ structure ↔ function relationships
• Experimental methods measure different physical properties
• Folding and binding are tightly coupled