Biochem Lecture #5 (Mestril) Flashcards
Explain the experiment that Dr. Alfisen did.
What were the conclusions of his experiment.
Dr. Alfisen added 8m urea to denature a RNAse, and then added beta-mercaptoethanol to reduce it and disrupt the disulfide bonds in the protein. This resulted in a loss of nature structure (aka denaturation) and a lost of enzymatic activity. Once he removed those agents slowly, he found that the protein refolded back into its native state and gained back its enzymatic activity (proving that it properly refolded and reformed S-S bonds).
CONCLUSIONS:
- The native structure is the most thermodynamically stable state for most proteins.
2. The native tertiary structure is determined by the primary structure of a protein.
What contains all of the necessary information to specify the 3D structure of a protein?
THE PRIMARY SEQUENCE of AA’s contains the message for 3D folding
What are the ways in which a protein can be denatured?
Proteins can be denatured depending on chemical enviornment:
Heat
Chemical denaturant (8M urea)
pH
high pressure
How does gibbs free energy relate to protein folding?
The confirmation of a protein is the lowest Gibbs free energy accessible to its sequence within a physiological time frame.
Protein wants to achieve LOWEST FREE ENERGY.
(Think about the funnel)
How does a polypeptide fold and maintain a stable native form?
HYDROPHOBIC BONDS
Rate the strength of the following from strongest to least: hydrophobic, covalent, hydrogen, ionic, vanderwalls
Strongest is the Covalent
Noncovalent is the second strongest category
Within noncovalent: Hydrophobic > Hydrogen > Ionic> van der waals
Explain the hydrophobic effect on protein folding
Water forms hydrogen bonds around hydrophobic solutes (reduces entropy…not good)
When two hydrophic ends are brought together (increases entropy which decreases Gibbs free energy)
Hydrophobic core of proteins and the lipid membrane interior both results from this… Bringing together hydrophobic pockets increases entropy and reduces gibbs free energy
Alpha helices and beta sheets are stabilized by?
HYDROGEN BONDS are important in alpha helices and beta sheets
Strongest of noncovalent forces is_____
Weakest of noncovalent forces is______
Strongest: Hydrophobic
Weakest: van der Waals
What kind of interaction happens when two aromatic rings are near each other?
Pi bond overlap
Why is it so much harder for proteins to fold “in vivo” aka inside the cell?
cells are super duper crowded
What are the predicted biochemical effects on the following due to macromolecular crowding:
Solubility
Diffusion rates
aggregation of partially folded proteins
effectiveness of chaperones
rates of folding
Cell crowding causes:
Decrease solubility of macromolecules
Decreased diffusion rates
Increased aggregation of partially folded proteins
Increased effectiveness of chaperones
Increased rates of folding for some proteins
Name the three protein types that help with folding
- Cis-trans-prolyl isomerases (interconvert cis and trans peptide bonds of proline residues)
- Protein disulfide isomerases (catalyze breakage and formation of disulfide cystine linkages)
- Chaperone proteins (prevent protein aggregation)
The cellular concentration of a protein is controlled by what?
Rate controlling step?
The cellular concentration of a protein is controlled by its rate of synthesis and of degradation.
The rate of denaturation is the rate controlling step
What are some common protein denaturants?
Urea, guanidine hydrochloride, SDS, strong acids or bases, high temperatures
