Chapter 3: 3.5 Analysis of Protein Structure Flashcards
True or False:
Each secondary structure has a unique set of bond rotation angles
True
What are the measurable angles of rotations in the secondary structure?
- Cα-C=O (ψ)
- Cα-NH (φ)
What is the unique set of bond rotation angles in α-helix?
- ψ: -50°
- φ: -60°
What is the unique set of bond rotation angles in β-sheet?
- ψ: +135°
- φ: -140°
Define:
Ramachandran Plots
Created by plotting the ψ angles of a protein’s amino acids on the y-axis and the φ angles on the x-axis
What does a Ramachandran Plot show?
Shows which kind of angle combinations are possible and which are not (steric hindrance)
ψ and φ angles define protein geometry, what can Ramachandran Plots define?
Protein structure
True or False:
Secondary structures show characteristic patterns on the Ramachandran plot
True
What is the speed of protein folding? What does this indicate?
- 10^3 - 10^-1 seconds
- Indicates that folding is directed
What did Anfinsen’s experiment discover?
Discovered that the primary sequence of proteins dictated the secondary and tertiary structure
What was the Anfinsen Experiment?
A series of experiments were conducted on Ribonuclease A
Angle combinations that are not possible are due to…
Steric hindrance
What is a protein’s folded state known as?
Native structure
Anfinsen Experiment rationale:
- What stabilizes a protein’s folded state?
- What should lead to the protein unfolding?
- How is this done? (protein unfolding)
- Disulfide bonds
- Reducing
- With β-mercaptoethanol
Anfinsen Experiment rationale:
When β-mercaptoethanol was applied, what happened?
Unfolding did not occur fully
Anfinsen Experiment rationale:
What had to be present in order for full protein unfolding to occur?
Urea or Guanidine
Anfinsen Experiment rationale:
Denaturing agents disrupt…
Non-covalent interactions
Anfinsen Experiment rationale:
How does urea break secondary structures?
Form H-bonds with the amino acid backbones
Anfinsen Experiment rationale:
To allow the protein to re-fold, how was urea and β-mercaptoethanol removed?
Dialysis
Anfinsen Experiment rationale:
Describe dialysis
Involves a semi-permiable membrane that allows some molecules to move from a place of high concentration to low concentration while others remain
Anfinsen Experiment rationale:
- What was the result from dialysis?
- What does this mean?
- Protein did not re-fold correctly
- Only 1% of activity was recovered
- Means there must be assisted folding
Unfolded proteins have…
- High free energy
- High entropy
What is entropy?
Measure of disorder
True or False:
There is only one possible folded state for a protein
False, there are many possible folded intermediate states
Which folded state has the lowest free energy?
The native structure
The energy difference between transition states and native strucutre…
Large
Transition states are the —– in the graph
Peaks
What does the large energy gap between transition states and native structure ensure?
Ensures protein unfolding does not occur
True or False:
Proteins smoothly fold
False, they can get “stuck” in energy wells
What assists in protein folding? How?
Chaperones
* Bind to exposed hydrophobic areas
What do chaperone proteins ensure?
- Ensure that improperly folded proteins don’t aggregate
- Mis-folded proteins are degraded by the proteosome
