chapter 9: Proteins and their folding states Flashcards
What happens when RNase A is placed in an 8M urea solution containing 2-mercaptoethanol
RNase is compeltely unfolded and its 4 disulfide bonds are reductively cleaved
When an RNase A is placed in an 8M urea solution containing 2-mercaptoethanol and then the urea is dialyzed away and exposing the resulting solution to O2 at pH of 8 what occurs?
It yields a protein that is ~100% enzymatically active physically indistinguishable from native RNase A.
The renaturation of RNase A demands what?
that its four disulfide bonds must reform.
What is the probability that the four disulfide bonds with its proper (native) mate will randomly reform?
Randomly reforming ONE cys residue with propoer mate is a 1/7 chance while reforming the second one is a 1/5 chance and etc…. 1/7) (1/5)(1/3)(1/1) = 1/105
Why is the native state of RNase A under physiological conditions most likely its thermodynamically stable conformation?
If the protein has a conformation that is more stable than the native state, conversion to it must involve such a large activation barrier so as to make it kinetically inaccessible.
what is “scrambled” RNase?
“scrambled” RNase A is if the RNase A is reoxidized in 8M urea so its disulfide bonds reform while the polypeptide chain is a random coil. Then after the removal of the urea the RNase A is expected to be only 1~ enzymatically active; this is “scrambled” RNase when it is only 1% enzymatically active
How can RNase be made fully active?
By exposing it to a trace of 2-mercaptoethanol, which over 10 h period catalyzes disulfide bonds interchange reactions until its native structure is achieved.
How can the time for renaturation of “scrambled” RNase A be reduced?
It can be reduced to ~ 2 min by using the enzyme protein disulfide isomerase (PDI)
How does PDI reduce the renaturation time of “scrambled” RNase A?
catalyzes disulfide interchange reactions; PDI contains 2 active site Cys residues which must be in the -SH form for the isomerase to be active. The enzyme evidently catalyzes the random cleavage and reformation of a protein’s disulfide bonds , thereby interchanging them as the protein progressively attains thermodynamically more favorable conformations
What happens if the RNase is placed in oxidizing conditions before the urea is removed then the urea is removed?
you achieve scrambled RNase A
Many “scrambled” proteins are _____ via action of PDI and are ______ affected by PDI in native state
“Scrambled” proteins are renatured via PDI but are unaffected by PDI in its native state
What may be the function of disulfide bonds?
may serve to hold protein in its otherwise unstable native state.
Insulin which has 2 polypeptide chains joined by disulfide bonds is inactivated by PDI but proinsulin which has a single polypeptide chain is activated by PDI. Why might that be?
Insulin is the most stable local conformation for this protein while proinsulin is the precursor for insulin. Insulin is only created after the disulfide bonds of proinsulin is created.
What is the function a C chain? Does it direct the folding of A and B chains?
No it simply holds together A and B chains while they form their native disulfide bonds.
What 2 observations suggest that C chain proteolytic excisions hold together A and B chains while they form their native disulfide bonds instead of directing their folding?
1) Under proper renaturing conditions, native insulin is obtained in 25-30% yield which increases to 75% when A and B chains are chemically cross linked.
2) Sequence comparisons indicate that mutations are accepted into the C chain at a rate 8 times that for the A and B chain.
