Block 1

Question 1

Name three key reasons why understanding protein function is important

Answer 1

Understanding biological processes, 2. Development of new drugs and pharmaceuticals, 3. Creation of new biomedical diagnostics

Question 2

How does a protein’s structure relate to its function?

Answer 2

A protein’s function is a direct result of its chemical and structural complexity

Question 3

What are the four levels of protein structure?

Answer 3

Primary, secondary, tertiary, and quaternary

Question 4

What is the primary structure of a protein?

Answer 4

The amino acid sequence

Question 5

How are amino acids classified?

Answer 5

Based on the nature of their side chains

Question 6

What are the characteristics of polar amino acids?

Answer 6

They can be charged or uncharged and are hydrophilic due to their ability to form hydrogen bonds with water

Question 7

What are the characteristics of non-polar amino acids?

Answer 7

They have no net charge and are hydrophobic as they do not bond with water

Question 8

Where are hydrophobic and polar amino acids typically located within a protein structure?

Answer 8

Hydrophobic amino acids form the core, while polar amino acids are on the surface

Question 9

What type of interactions are key to holding proteins together?

Answer 9

Non-covalent interactions, such as electrostatic and hydrophobic interactions.

Question 10

What are torsion angles?

Answer 10

-Angles that show how the protein backbone twists.
Phi (φ): Twist at N–Cα (Nitrogen - central carbon)
Psi (ψ): Twist at Cα–C
Omega (ω): Twist at peptide bond (usually flat, 180°)
- The N–Cα and Cα–C bonds are single bonds, so they can rotate → this gives us the φ (phi) and ψ (psi) angles.
-But the C–N (peptide bond) between amino acids has partial double bond character. it acts as a doubke bond so rotation cant occur hence why it makes the backbone flat at than point

Question 11

Which torsion angles provide conformational flexibility and determine secondary structure?

Answer 11

Ψ and Φ

Question 12

What does a Ramachandran map define?

Answer 12

it defines the allowed combinations of ψ and φ angles, showing the possible conformations of a protein
-Because atoms have size and can’t bump into each other (called steric hindrance), not all φ and ψ angles are possible. The map shows which ones are allowed.

📈 What it looks like:
It’s a graph with:

φ (phi) on the x-axis

ψ (psi) on the y-axis

The shaded regions show where the angles can exist without causing clashes in the structure.

Common regions correspond to structures like:

Alpha helices

Beta sheets

Question 13

What are the most common types of secondary structures in proteins?

Answer 13

α-helices= Coiled, spiral shape held by H-bonds within one strand.
Beta Sheet: Flat, sheet-like shape held by H-bonds between strands.

Question 14

What are motifs?

Answer 14

Motifs are simple, short patterns of secondary structure (like alpha helices and beta sheets) that appear again and again in different proteins.
-help form the proteins structure

Question 15

What stabilises the structure of an α-helix?

Answer 15

-Hydrogen bonds, van der Waals interactions and non-polar interactions

Question 16

What are the characteristics of β-sheets?

Answer 16

-: They are formed by H-bonds between peptide chains,
- R-groups alternately above and below the plane,
-can be parallel or anti-parallel

Question 17

What are β-turns and what are their typical features?

Answer 17

-Often found between strands of beta sheets
Short turns that reverse the direction of the peptide chain, often containing cis-proline at the 2nd position and glycine at the 4th position, with a hydrogen bond between residue i and i+3

Question 18

What is the function of loops in protein structure?

Answer 18

Connect secondary structure elements, typically hydrophilic and on the surface, often form binding or active sites, provide dynamism and flexibility
-enable large proteins to fold into compact structures

Question 19

What are domains in the context of protein structure?

Answer 19

Combinations of motifs
-Larger functional units in a protein.
-Can consist of multiple motifs that come together to form a self-stable structure.
-Can fold independently and often have their own specific functions (e.g., binding DNA or catalyzing reactions).

Question 20

How are protein domains classified?

Answer 20

Purely α-helical, purely β-stranded, or combinations of both (α+β or α/β )

Question 21

What is quaternary structure?

Answer 21

The interaction of at least two polypeptide chains

Question 22

What are homo-oligomers?

Answer 22

Multiple copies of the same polypeptide chain interacting

Question 23

What are hetero-oligomers?

Answer 23

Multiple copies of different polypeptide chains interacting

Question 24

Why is quaternary structure important?

Answer 24

Generating large stable structures, bringing catalytic sites together, increasing stability, facilitating cooperativity and allostery, and reducing exposed surface area

Question 25

Name some functions of membrane proteins.

Answer 25

Enzymes, signal transducers, transporters, and cell-cell recognition molecules

Question 26

What are the main properties of proteins that are exploited for purification?

Answer 26

Solubility, binding specificity, ionic charge, and molecular size

Question 27

Name three methods of cell disruption (cell lysis) used to release proteins for purification.

Answer 27

- Mechanical disruption (sonication, high-pressure. Ecoli -homogenisation (bead milling), in yeast and other tougher cells -chemical disruption (detergents), and enzymatic disruption (lysozyme), ecoli and yeast

Question 28

What is the principle of ammonium sulphate precipitation?

Answer 28

Add ammonium sulfate to the protein solution. -Proteins precipitate at different concentrations of ammonium sulfate due to differences in solubility. -Centrifuge to separate precipitated proteins (pellet) from the supernatant. Fractionate the protein mixture by controlling the salt concentration, isolating specific proteins. -the proteins that are more soluble in the solution will remain in the supernatant (the liquid part), while the less soluble proteins will precipitate out and form a pellet.

Question 29

What is the basis of affinity chromatography?

Answer 29

Separation based on binding affinity between a protein and a specific ligand. This method is often used for recombinant proteins and achieves very high levels of purity. 3 steps: Loading: Protein mixture is added to a column with a ligand attached to the resin. The target protein binds to the ligand. Separation: Wash the column to remove unbound proteins. Elution: Add an elution buffer to release the bound protein by disrupting its interaction with the ligand.

Question 30

What are the two main types of ion exchange chromatography?

Answer 30

Anion exchange (using positively charged resins to bind negatively charged proteins) and cation exchange (using negatively charged resins to bind positively charged proteins) -on Exchange Chromatography: Loading: Protein mixture is applied to a column with charged resin (positive for cation exchange, negative for anion exchange). Proteins bind based on charge. ( opposite charge bind, same charge flow through) Separation: Wash the column to remove unbound proteins. Elution: Use an elution buffer (higher salt or changing pH) to release proteins based on their charge.

Question 31

What are the two main types of ion exchange chromatography?

Answer 31

Anion exchange (using positively charged resins to bind negatively charged proteins) and cation exchange (using negatively charged resins to bind positively charged proteins)

Question 32

How does size exclusion chromatography (SEC) separate proteins?

Answer 32

How it works: Proteins are separated based on size using a column with porous beads. Larger proteins elute first because they don’t enter the beads and move faster. Smaller proteins elute later because they enter the beads and take longer to travel through the column.

Question 33

How does hydrophobic interaction chromatography work?

Answer 33

-Proteins with hydrophilic and hydrophobic regions are applied at high salt conditions. -the salts exposed the hydrophobic regions of proteins -hydrophobic regions of proteins bind to the ligand. -When the salt concentration is reduced, proteins are eluted in order of increasing hydrophobicity

Question 34

Why are detergents used in membrane protein purification?

Answer 34

Detergents are amphipathic molecules, meaning they have both hydrophilic (water-attracting) and hydrophobic (water-repelling) regions. When added to a membrane protein solution, detergents disrupt the lipid bilayer by inserting their hydrophobic tails into the membrane, breaking it apart. The hydrophilic heads remain in the surrounding aqueous environment. This process forms micelles, which are aggregates of detergent molecules that surround the membrane proteins. The hydrophobic regions of the proteins are trapped inside the micelle, while the hydrophilic regions interact with the water, keeping the protein in solution and stabilizing it in a mildly denaturing environment. This prevents the protein from aggregating and maintains its functional state.

Question 35

co expression of muliprotein complexes in bacteria

Answer 35

A multiprotein complex is a group of two or more proteins that work together as a functional unit. In bacteria, these complexes are made by co-expressing all the protein subunits at the same time. This is done using multiple plasmids or a single plasmid with multiple promoters, allowing simultaneous expression. Co-expression ensures subunits are made in the right amounts and can assemble correctly into the complex. Sometimes, chaperones are co-expressed to help with folding and assembly.

Question 36

how do we stabilize multicomplex proteins?

Answer 36

- need to be stabilzed as they are large -used fractionation methods such as... Sucrose density gradient and CsCl gradient

Question 37

recombinant protein expression

Answer 37

can use multiple different expression systems. bacteria- Ecoli (not reliable) but fastest and cheapest yeast, insect cells, mammalian cells all very reliable

Question 38

how do we use bacterial promotors for inducible expression of proteins

Answer 38

Grow bacteria in log phase (active cell division). Add inducer (e.g., IPTG) to bind to the promoter and remove the repressor, turning on gene expression. Shift temperature (e.g., 30°C or 18°C) to help protein folding. Harvest and extract the recombinant protein after a few hours.

Question 39

what methods do we use to study protein structure?

Answer 39

-microscopy(light,electro or x-ray,) -NMR -cyro-electron microscopy

Question 40

circiular Dichroism (CD)

Answer 40

Circular Dichroism is a technique used to study the structure of proteins, especially their secondary structure (like alpha helices and beta sheets). It works by shining circularly polarized light on a protein solution. Different structures in the protein absorb this light differently, which creates a signal. By analyzing this signal, we can tell if the protein is mostly alpha helix, beta sheet, or random coil—helping us understand its folding and stability.

Question 41

SEC multiple angle light scatttering (SEC MALS)

Answer 41

DLS is a technique used to measure the size of particles or proteins in a solution. It works by shining a laser on the sample. As the particles move (diffuse), they scatter the light in different ways. The instrument measures these changes over time. From this, we can calculate the average size of the particles and check if the protein sample is monodisperse (uniform) or aggregated.

Question 42

isothermal calorimetry (ITC)

Answer 42

ITC is a technique used to study binding interactions between molecules, like a protein and its ligand. It works by measuring heat changes when one molecule is gradually added to another. If they bind, heat is either released or absorbed. From this, we can learn how strong the binding is (affinity), how much of it occurs (stoichiometry), and the thermodynamics (enthalpy and entropy) of the interaction — all without needing labels or modifications.

Question 43

purification summarised

Answer 43

🧪 Protein Purification Methods Used to isolate your protein from a mixture: Affinity Chromatography – Grabs your protein based on a specific tag or binding. Ion Exchange Chromatography – Separates by charge. Size Exclusion Chromatography (SEC) – Separates by size. Ammonium Sulfate Precipitation – Uses solubility to separate proteins. 🧫 Expression Systems Used to make your protein (especially recombinant proteins): Use bacteria (like E. coli) to express single or multiple proteins. Add inducers (like IPTG) during growth to control when the protein is made. 🧴 Membrane Protein Help Special strategies for tricky proteins: Detergents are used to solubilize membrane proteins and keep them stable. 🧲 Characterization Techniques Used to study your protein’s structure, size, or interactions: Circular Dichroism (CD) – Tells you about secondary structure (e.g. alpha helix). Dynamic Light Scattering (DLS) – Tells you the size and whether your protein is aggregated. Isothermal Titration Calorimetry (ITC) – Tells you how your protein binds to something else (and how strong that binding is).

Question 44

The steps of purification of a protein

Answer 44

xpress → Solubilize (if needed) → Purify → Study