lecture 24 - globular protein folding

Question 1

explain general characteristics of globular protein folding

Answer 1

diffucult to observe:

• can be fast
• may start while proteins are still being synthesized
• not a random process (not all possible phi and psi angles are explored for each aa)

Question 2

explain (generally) how we study globular protein folding (3 steps)

Answer 2

(1) apply heat
(2) adjust pH
(3) apply chaotropic agents

Question 3

explain the process of heat degradation for observing globular proteins

Answer 3

• abrubt loss of structure around 50% native fold suggests a cooperative process
• relates to the fact that there are large #s of weak interactions that stabilize proteins

Question 4

explain the process of adjusting pH to observe globular protein folding

Answer 4

• apply an extreme pH (=2 or >/=12)
• affects the ionization states
• results in a net charge
• causes electrostatic repulsion
• disrupts hbonding and salt bridges

Question 5

explain the use of chaotropic agents to observe globular protein folding

Answer 5

• Urea (8M) and Guanidinium salts (6M)
• form hbonds to protein disrupting the secondary protein structure
• stabilize and solubilize the unfolded state (no aggregation)
• disrupt the hydrophobic core

Question 6

explain the process of refolding

Answer 6

• elevate temperature and then reduce temperature OR
• use urea to unfold then slowly remove urea by dialysis (have protein w urea and buffer w no urea so it diffuses out)
• either allows the system to reach equilibrium slowly
• allows protein to refold

Question 7

explain the Anfinsen experiment

Answer 7

-Ribonuclease A (124 aa, 8 cys residues, 4 disulphide bonds) was denatured with urea, resulting in reduced disulphide bonds (and free cys residues)
-then ran two experiments with the unfolded RNase A:
(1) removed urea which allowed refolding, then oxidized
the protein, which allowed disulphides to form
-resulted in 100% activity (disulphide bonds are not involved in folding)
(2) oxidized to allow disulphides to form, then removed urea to allow for refolding
-resulted in ~1% activity
-~105 ways to form disulphide bonds randomly between 8 cys residues (7x5x3)

Question 8

what were the conclusions of the Anfinsen experiment?

2

Answer 8

• primary structure determines the 3D fold and therefore the function of the protein
• disulphides form after proteins are folded to help stabilize the folded state but do not help the folding process

Question 9

explain the folding process theoretically

Answer 9

-DYNAMIC property of proteins
-proteins fold to form a small # of low-energy conformations
-this energy of the system can be reperesented by a funnel, where the diameter represents the number of conformations:
-at the top - high energy and many conformations
-as funnel narrows = decreasing energy and decreasing number of conformations
-at the bottom, several (interchangable) low energy conformations
=the native (functional) state of the protein

Question 10

explain the folding process in vivo

Answer 10

• proteins start folding as they exit the ribosome
• chaperones aid folding and refolding
• enzymes aid proper formation of bonds and conversion of necessary molecules

Question 11

how do chaperones aid folding and refolding?

Answer 11

• by providing an enviroment to allow folding inside a chamber
• preventing aggregation via exposed hydrophobic regions

Question 12

what is disulphide isomerase?

Answer 12

enzyme that assists w correct disulphide formation

Question 13

what is peptide prolyl cis-trans isomerase?

Answer 13

enzyme that catalyzes the interconversion of cis and trans prolines