Lecture 6 - Protein Folding Part II

Question 1

Define and Explain the difference between Primary and Secondary Protein Structures.

What are the main stabilizing factors in a secondary bond?

Answer 1

Primary Structures - Amino Acids Linked together by Peptide Bonds
Secondary Structures - Polypeptide Proteins can be folded into either Alpha Helices, Beta Sheets, Omega Loops (also called Beta Turns, or Hairpin Turns)

Stabilizing Factors Include: 1. (Short Range Repulsion), 2. (H-Bonds), 3. (Van Der Waals forces), 4. (Ion Pairs and Salt Bridges)

Question 2

• Define Tertiary Structures and
• State their stabilizing factors
• Define Quaternary Structures

Answer 2

• Water soluble proteins that fold into compact structures with nonpolar cores
  – Stabilizing factors: Disulfide bonds (S-S); hydrophobic properties
• Polypeptide chains can assemble into multisubunit structures

Question 3

Where are disulfide bonds usually found in polypeptide chains?

Answer 3

Extracellular proteins mostly have S-S bonds; most of the intracellular proteins usually lack S-S bonds

Question 4

Describe some characteristics of protein folding

Answer 4

• Tertiary Structure can easily be denatured by breaking S-S bonds
• The unfolding process is all-or-none. Denature one part, and the rest unfolds, and during denaturation, the protein very briefly becomes a molten globule intermediate

Question 5

Describe the Chou Fasman method

Answer 5

The Chou Fasman method is a documented approximation of the ratio of alpha helices vs. beta sheets in every type of amino acid, as well as their number of reverse turns (in secondary structure)

Question 6

Define Chou Fasman frequency (f)

Define Chou Fasman Propensity/Natural Inclination (P)

Answer 6

f = (Amino Acids in the protein that are in Alpha Helix mode)/(Total Amino Acids)

P = (Frequency of aa(i)) / (All aa(i)) / (All Amino Acids)

…yeah, may wanna review this one

Question 7

Name 3 examples of Physical Conditions that can Denature Proteins.

Name 3 examples of Chemicals that can Denature Proteins

Answer 7

Physical: 
1. Heat (Like frying an egg)
2. Extreme pH
3. Heavy Agitation (Shaking it too hard)
Chemical:
1. Detergents (Such as SDS)
2. Chaotropic Agents (Urea, Guanidine Hydrochloride)
3. Organic Solvents (TCA)

Question 8

Name 5 methods used to determine and analyzed the conformation of proteins

Answer 8

1. Turbidity
2. CD (Circular Dichroism)
3. UV Absorption
4. Fluorescence
5. Biological Activity

(Will need to go back at some point and figure out what some of these are)

Question 9

How does the reducing agent B-Mercaptoethanol denature proteins?

Answer 9

Reducing Agent targets disulfide bonds for it’s own use, allowing protein to unravel. (See Slide 13 for diagram and description)

Should know that B-mercaptoethanol is OH-CH2-CH2-SH

Question 10

Name the 3 types of Accessory Proteins used in Protein Folding

Answer 10

1. PDI - Protein Disulfide Isomerases
2. PPi - peptidyl prolyl cis-trans isomerases
3. Molecular Chaperones

Question 11

Describe the 5 types of Molecular Chaperones

Answer 11

1. HSP70 Family - ATP driven protein that can reverse misfolded proteins, and helps refold and unfold trafficked proteins
2. Chaperonins
3. HSP90 - Signal Transduction Protein
4. Nucleoplasmins
5. Small HSPs (Such as alpha-crystallin)

Note that mitochondria have different shaped Hsp60 and Hsp70 molecules than the rest of the cytosol

Question 12

What is the purpose of Protein Disulfide Isomerase (PDI)?

Answer 12

Rearranges non-native sulfide bonds into their correct “position” in secondary structures.

(Will Very Likely Need to Know Mechanism Described on Slide 15)
I don’t totally get it yet, but it changes the 1-2, and 3-4 sulfide bonds into the proper 1-3, and 2-4 bond shape.

Question 13

What is the purpose of Peptidyl prolyl cis-trans isomerase (PPI)

Answer 13

Switches a peptide bond from trans form to cis form and vice versa with the right enzyme…I think?

Question 14

What is the purpose of Molecular Chaperones?

Answer 14

• Essential proteins that bind to unfolded and partially folded polypeptide chains
• They prevent the improper association of exposed hydrophobic segments
• Non-native folding, polypeptide aggregation, and precipitation will not occur
• They allow misfolded proteins to refold into their native conformation

Question 15

Describe the 2 largest classes of chaperones.

Answer 15

1. Hsp70 family - Helps prevent premature folding
2. Chaperonins (with large multiple subunit proteins)
   - Hsp60 proteins (GroEL)
   - Hsp10 proteins (GroES ) (co-chaperone)

(And I don’t know what the hell’s going on in slide 19)

Question 16

Know the Structure of Chaperonin 10 and Chaperonin 60

Answer 16

Alright, so I think the chaperonin is divided into two pieces: GroES, and GroEL. (Also called chaperonin 10 and 60 respectively)
GroES is the “lid” to the chaperonin. GroEL is the chamber (there’s actually 14 GroELs that make up the chamber.
GroEL is divided into 3 parts from top to bottom (Apical, Hinge, And Equatorial), and the GroELs are connected by Ring Contacts.

Question 17

Describe the Mechanism of Chaperonins in protein folding

Answer 17

1. You’ve got a cap and a cylinder. Cap comes off, unfolded polypeptide goes in.
2. The cap goes back on and the cylinder rearranges it’s shape so that it creates a hydrophilic environment to fold the polypeptide
3. Cap comes off, correctly folded protein comes out. Presumably, the protein is properly folded through protein-protein interactions, but nobody’s quite certain.