4-7 PROTEIN DENATURATION, RENATURATION AND FOLDING
Denaturation

Denaturation of proteins is caused by _______________ changes or _______________
treatments that disrupt the __________ conformation.
o
This also causes the protein to lose its _______________ activity.
o
Some denature and _______________ completely, while others may retain considerable
_______________ structure.

Proteins commonly denature upon _______________, resulting in the loss of secondary and
tertiary structure.
o
The destabilization of just a few __________ interactions leads to almost complete loss
of native conformation.

Proteins can also be denatured by …
o
_______________: Chemicals like urea and guanidinium chloride allow water molecules
to disrupt the hydrophobic interactions in the interior of proteins.
o
_______________: Hydrophobic tails of these (like SDS) denature proteins by
penetrating the protein interior and disrupting hydrophobic interactions.

The presence of _______________ bonds makes the protein less susceptible to unfolding.
o
Thiol reagents like _______________ can be added to a denaturing medium in order to
reduce disulfide bonds.
Renaturation

Christian B. Anfinsen and his coworkers studied the renaturation pathway of ribonuclease A.
o
Added 8 M urea and 2-mercaptoethanol to ribonuclease A and caused the complete loss
of _______________ structure and _______________ activity.
o
When the 2-mercaptoethanol is removed, the –SH groups re-formed the four disulfide
bonds _______________, in 105 different possible combinations, but…
o
When both urea and 2-mercaptoethanol are removed simultaneously and the protein is
exposed to air, ribonuclease A spontaneously regains its _______________
conformation, its _______________ set of disulfide bonds, and full _______________
activity!
Denaturation and renaturation of ribonuclease A:

Some proteins fold incorrectly and form incorrect disulfide bonds. Anfinsen discovered an
_______________ that can catalyze the reduction of these incorrect bonds and allow the
misfolded protein to refold into the intended native conformation!

Anfinsen won the 1972 __________ _________ in Chemistry for his work on the refolding of
proteins.
Protein Folding

Polypeptides are synthesized in the cell by a translation complex within ribosomes.

As the polypeptide emerges from the ribosome, it __________ into its __________ conformation.
This conformation has the __________ possible energy, making it the most __________
conformation possible.

Proteins fold through ____________________ of folding – where the formation of one part of a
structure leads to the formation of the remaining parts of the structure.

Folding is extremely __________ - taking less than a second.
o
The polypeptide collapses upon itself due to the hydrophobic effect, causing elements of
_______________ structure to form.
o
Subsequent steps involve the rearrangement of the backbone chain to form characteristic
__________, and finally, the native conformation.
o
The _______________ of protein folding is one of the most challenging problems in
biochemistry. No actual protein-folding pathway has yet been described in detail.

In general, there are four forces that stabilize protein structure during folding:
1. _______________ effect: Water forces the nonpolar side chains to associate with one
another, causing the protein to _______________ into a more compact globule.
a. This decrease in disorder (or _______________) is the driving force for
protein folding.
2. _______________ bonding: These interactions in  helices and  sheets are the first
to form, giving rise to secondary structure. These also form between these secondary
structures, giving rise to _______________ structure and eventually the final
__________ structure.
3. _____ _____ __________ Interactions: Another name for London dispersion forces,
the cumulative effect of all of these nonpolar forces contribute significantly to the
stability of a protein.
4. __________-__________ Interactions: These may occur between oppositely-charged
side chains, and contribute minimally to the stability of a protein.
Molecular Chaperones

Folding is not a __________ search for its native conformation. It is a cooperative, sequential
process in which a few initial interactions lead to the formation of subsequent structural features.

Folding is assisted by molecular _______________, which…
o
Increase the __________ of folding.
o
Prevent the formation of _______________ folded
intermediates.
o
Prevent protein subunits from aggregating incorrectly
and _______________ before they are completely assembled.

Read pages 113 (2nd full paragraph) through the end of the section on page 115 to learn more
about molecular chaperones.