lec12-13 Flashcards
What is the native state of a protein?
The folded state, which is the most stable, lowest energy form (negative delta g).
What causes protein denaturation?
-High temperature
-Extreme pH
-Chaotropic agents (8M urea, 6M guanidinium chloride)
-Detergents (disrupt hydrophobic and polar interactions)
How can a denatured protein refold?
By removing denaturants or restoring proper conditions (e.g., correct temperature, pH).
denatured proteins are usually insoluble cuz the hydrophobic regions are exposed.
How do reducing agents like β-mercaptoethanol (β-ME) and DTT affect proteins?
They break disulfide bonds, destabilizing the protein and contributing to denaturation. As they reduce disulfide bonds, they themselves get oxidized and form disulfide-bonded dimers. (dimer is a molecule made of two subunits bonded together eg) in this case 2 B-ME bonded by dislufide bonds)
2 Types of Denaturants and Examples
- Chaotropic agents – disrupt non-covalent interactions (e.g., 8M urea, 6M guanidinium chloride)
- Reducing agents – break covalent disulfide bonds (e.g., β-mercaptoethanol (BME), dithiothreitol (DTT)
What did Anfinsen’s experiment show about protein folding?
-Denaturation: Incubating ribonuclease in 8M urea + β-mercaptoethanol unfolds the protein.
-Renaturation: Slowly removing urea first, then β-ME allows the protein to refold.
-Performed in vitro (in a test tube, outside a cell), proving that the amino acid sequence alone determines the correct 3D structure of a protein.
What is the difference between in vitro and in vivo protein folding?
-In vitro (test tube): Protein starts as a denatured random coil and folds into its native state without cellular help.
-In vivo (inside a cell): Proteins fold during synthesis on the ribosome, sometimes with chaperone assistance.
-Folding happens within seconds or less in both cases.
levinthal paradox
-most small proteins fold spontaneously in milliseconds or microseconds; thus it cant sample all possible conformations to get to the correct native form
-in a protein there are 3 possible conformations per residue (3^# of amino acids=# of different conformations)
-The enormous difference between the calculated and the actual folding time is
referred to as the Levinthal paradox
how to calculate theoretical folding time
of conformations:
3^(# of amino acids)=# of possible conformations
theoretical time:
- assume 1 conformation can form every 10^-13 seconds (100 femtoseconds)
eg) then 5 x 10^47 x 10-13 s = 1.6 x 1027 years to correctly fold a protein
protein folding theory: a folding funnel
The protein folding funnel represents the energy landscape of protein folding, showing how an unfolded protein (high energy, high entropy) progressively folds into its native state (N), which has the lowest free energy.
States A & B: Unfolded conformations with high energy.
Intermediate states: Temporary, partially folded structures that proteins pass through.
Native structure (N): Final, stable, lowest-energy state.
Folding follows a descent in energy, avoiding misfolding traps along the way.
What are the three proposed models of protein folding?
Hydrophobic Collapse Model – The protein rapidly collapses due to hydrophobic interactions, forming a compact structure. Secondary and tertiary structures then refine within this collapsed state.
Framework Model – Secondary structures (helices and sheets) form first, then they assemble into the final tertiary structure.
Nucleation Model – A small, stable nucleus of secondary structure forms first, then the rest of the protein folds around it in a cooperative manner.
✅ All models lead to a folded protein with the lowest Gibbs free energy (ΔG = ΔH - TΔS)
How do DTT and BME reduce disulfide bonds in proteins? and which is the stronger reducing agent
They donate electrons to break the –s–s– bond, forming two –sh groups while oxidizing into their own disulfide dimer. dtt is the stroger one cuz it can form a dimer with itself as it has two S unlike bme
What does the protein folding funnel illustrate?
Proteins do not randomly sample all conformations but follow an energy landscape, moving through folding intermediates to reach the lowest energy native state.
What are two examples of chaperone proteins and their functions?
Hsp70 binds unfolded proteins to prevent aggregation and uses ATP to assist folding, while GroEL-GroES acts as a folding chamber where misfolded proteins are enclosed and refolded using ATP.
How does the GroEL-GroES chaperonin assist protein folding
GroES and ATP bind to GroEL, trapping an unfolded protein inside. ATP hydrolysis triggers a conformational change, allowing the protein to fold and be released, while a new unfolded protein enters the cycle.
what lengths of amino acids in a domain have difficultises in folding and what exactly is difficult?
domains tend to have ~40 to 200-350 amino acids
- fewer than 40 amino acids - difficult to fold stably
- more than 300 difficult to fold co
What is a protein domain?
🧩 A protein domain is a distinct structural and functional unit within a protein.
Acts like a LEGO piece, folding independently and performing a specific task.
Examples:
Catalytic domain – helps with chemical reactions (e.g., kinase domain).
Binding domain – attaches to other molecules (e.g., DNA-binding domain in transcription factors).
Proteins can have multiple domains, like tools on a Swiss Army knife!
structure of globular proteins (domains)
domains tend to have ~40 to 200-350 amino acids- fewer than 40 amino acids - difficult to fold stably- more than 300 difficult to fold correctly
* a single domain is typically made of a single stretch
of primary sequence
* the interior consists almost entirely of non-polar
residues
* both polar and non-polar residues on the outside
What are the differences between fibrous and globular proteins?
🌀 Fibrous vs. Globular Proteins
Fibrous Proteins – elongated, structural, insoluble (ex: collagen, α-keratin).
Globular Proteins – compact, spherical, functional, soluble (ex: myoglobin, RNase A, chymotrypsin).
Key Difference – fibrous proteins provide strength/support, globular proteins catalyze reactions or transport molecules.
❓ What are the structural classifications of globular proteins?
🌀 Globular Protein Structures
Predominantly α-helix – tightly packed α-helix bundles (ex: myohemerythrin).
Predominantly β-sheet – β-sheets form sandwiches or barrels (ex: prealbumin, immunoglobulin).
Mixed α-helix & β-sheet – both structures present, forming barrels or twisted sheets (ex: pyruvate kinase, hexokinase).
Key Concept – structure determines function in proteins!
❓ What are examples of quaternary protein structures?
Homodimer – two identical subunits (ex: Cro protein of bacteriophage λ).
Heterotetramer – four subunits, two α and two β (ex: human hemoglobin (α₂β₂)).
Key Concept – quaternary structure involves multiple polypeptide chains working together!
What is an example of a quaternary structure in bacteria?
eg)🦠 Bacterial Pili (Type IV Pilus)
Hair-like filaments on bacterial surfaces.
Made of thousands of pilin protein subunits.
Functions in motility, adhesion, and DNA uptake.
Example: Vibrio cholerae and Neisseria gonorrhoeae use pili for infection.
❓ What are examples of covalent modifications of amino acids in proteins?
addition of hydroxyl groups to prolines by the enzyme prolyl hydroxylase à hydroxyproline – (recall) in collagen to stabilize triple helix fibers - scurvy is caused by Vitamin C (ascorbic
acid) deficiency, which is necessary for the prolyl hydroxylase activity - results in abnormal
collagen fibers and tissue abnormalities
*addition of a second carboxylate (COO-) group to the g-carbon of glutamate ® g
carboxyglutamate - prothrombin requires g-carboxyglutamate for proteolysis, blood clotting
* can also have addition of carbohydrates, fatty acids
*phosphorylation of serine, threonine or tyrosine hydroxyl groups - reversible regulation
*acetyl groups [CH3C(=O)NH–] are often added to the amino terminus of proteins to make
them more resistant to degradation; acetylation of lysines on histones to remove their
positive charges
Which enzyme is responsible for the addition of hydroxyl groups to prolines in collagen?
prolyl hydroxylase
What deficiency causes scurvy due to its role in prolyl hydroxylase activity?
vit c
What is the function of γ-carboxyglutamate in proteins?
Proteolysis and blood clotting
Phosphorylation commonly occurs on which amino acids?
Serine, threonine, and tyrosine
What is the role of acetyl groups added to the amino terminus of proteins?
Making proteins more resistant to degradation
Which of the covalent modifications involves the addition of a second carboxylate group to glutamate?
γ-Carboxyglutamate formation
Which covalent modification removes positive charges from lysines on histones?
Acetylation. this makes them more resistant to degredation
