Protein Folding Flashcards
- What are the two denaturing agents Haber & Anfinsen used in their experiments with ribonuclease?
- What does each agent do?
- Urea: disrupts non- covalent bonds (ionic or hydrogen bonds). It is a chaotropic salt.
- BME: reduces disulfide bonds.
Both disrupt tertiary structure.
Describe the structure of ribonuclease A, the protein used in Haber & Anfinsen’s experiments
- It has three alpha helices and a central beta sheet
- Four disulfied bonds.
What are three lessons which were taken from the Haber & Anfinsen experiment?
- The denatured state can be very different from the native protein state.
- Denatured proteins can spontaneously fold to form the native state given the right solution conditions.
- Denatured proteins can misfold to form non-native structures given improper solution conditions.
What do denaturants allow us to do?
Give some examples of denaturing.
Control the relative amount of folded and unfolded states present in a solution.
- Chaotropic salts
- Reducing agents
- Heat
- pH
How do Chaotropes work?
reduce the energy penalty normally associated with exposing hydrophobic bonds by interacting with both polar and non-polar atoms.
What happens if a protein with a X-Proline bond has the incorrect isomer?
It must go through a slow isomerization to reach the correct form so that it can fold properly.
What are some folding situations of multidomain proteins?
- Each domain folds independently
- Slow association of folded domains: This requires small adjustments to get over activation barriers. These proteins may aggregate with other partially folded molecules.
- Interdependent/ cooperative domain folding
- Oligomeric proteins: monomers need fold into position and then, or concurrently be oligomerized together.
What are some generalizations in protein folding?
- Small proteins fold the same way in vivo and in vitro
- Membrane proteins are weird
- large proteins consisting of multiple domains are more complex, but their individual domains can still fold like small proteins.
What is the main concept behind transverse urea gradient gel electrophoresis?
Unfolded proteins at higher concentrations of urea will migrate more slowly than the relatively compact folded proteins.
What is the wavelength of Far-UV and Near-UV in circular dichroism
Far-UV: 190-230 nm
Depends on arrangements of peptide bonds in secondary structure
Near-UV: 280 nm
Depends on arrangement of aromatic residues like Phe, Tyr, and Thr
What is the emission wavelength maximum of buried, surface and exposed Tryptophan residues? Which indicates the most unfolded protein?
Buried - 320-330 nm - native
Surface - 340 nm
Exposed -350 nm - unfolded
What is a complication of using ANS to measure exposed hydrophobic groups/ presence of non-native protein states?
ANS can perturb the conformation of a protein upon binding to a hydrophobic residue.
Hydrogen Deuterium Exchange
Uses the exchange between hydrogens of amide backbone. Amides in disordered regions of a protein, like loops, exchange faster than those that are more ordered (alpha-helices)
- incubate protein in D2O for a set time
- Quench exchange by shifting to a low pH and low temperature (freeze reaction)
- Digestion with proteolysis creates peptide fragments.
-Separate peptides in chromatogram
- Measure the m/z of each peptide
What is the structure of human growth hormone? How does this related to HDX analysis?
- Four-helix bundle
Where is HDX most likely to occur?
- amide groups in loop regions are more likely to experience deuterium exchange than amide groups with hydrogen bonds or buried in the centre of the protein.
