How to calculate propensity for a given 2* structure
P= frequency/all AAs
Ex: P of beta-sheets=0.23/19 AA long protein
A given peptide is 20 amino acids long. 5 of those amino acids form an alpha-helix. What is the frequency of alpha-AAs?
A given peptide has an alpha helix frequency of 0.25. Given that the protein is 13 amino acids long, what is the propensity for alpha-helices?
P = freq/all AA
What two broad mechanisms can be used for protein denaturation?
Chemical and Physical
Used to break most hydrogen bonds and hydrophobic interactions
Changes to pH produce protein _____
Aside from changes to pH and addition of heat, what is the third thing to denature proteins?
How do detergents denature proteins?
There are three “parts” to a detergent: (-) charged polar head, fatty acid tails, and a (+) ion to balance the (-) polar head. SDS acts to disrupt non-covalent bonds
Acts to denature proteins by interfering with hydrogen bonds.
How do organic solvents denature proteins?
The electronegative oxygen in solvents functions to disrupt hydrogen bonding (denatures 2* structure)
How do reducing agents denature proteins?
-S-S- bonds are part of the 2* structure; the reducing agents reduce disulfide bonds to thiols (from -S-S- to -SH and HS-)
Aromatic rings absorb UV light at ____
Peptide bonds absorb UV light at _____
How does circular dichromism (CD) act to determine protein structure?
Amino acids will rotate polarized light clockwise (+) or counter-clockwise (-)
Describe fluorescence analysis of protein structure
Aromatic AAs (W, F, Y) all have natural fluorescence. This is measured to determine the presence of aromatic AAs.
Corrects misfolding and adds -S-S- bonds to newly synthesized proteins
PDI - protein disulfide isomerase
Reduces steric hindrance by moving large groups from cis to trans position
PPI - peptidyl prolyl cis-trans isomerase
Reverses mis-folded proteins; acts to unfold/refold proteins that are being trafficked; utilizes ATP
Mitochondria specific HSPs
Describe the GroEL/ES system
1) Unfolded protein attaches to GroEL cavity
2) 7ATP bind to GroEL and GroES cap binds
3) Protein undergoes new folding with loss of 7 Pi
4) 7 additional ATP bind to “swell” GroEL, GroES cap dissociates
5) Newly folded protein released
How to calculate alpha-helix frequency
F-alpha= (AA-alpha/all AAs)
Ex: F-alpha= (13 AA-alpha/19 AA long protein)