Things that denature polypeptide chain: Heat, extreme pH change, denaturants (urea,
Urea allows hydrophobic groups to be exposed
Hydrophobic collapse is a fancy way of saying all hydrophobic residues cluster as far away
from water as they can rapidly, and it’s the most important physical property of polypeptide
chain that allows folding.
This first step is called molten globulin.
It is very fast.
protein folds and domains are formed.
At beginning of unfolded stage you have high entropy level, and difference of entropy is no
more than a few H bonds.
Protein starts to collapse, and entropy starts to minimize.
globular state, you get to low level of entropy.
2 major forms of chaperones: Hsp 70 and DnaJ
Chaperones use ATP hydrolysis to help protein form into native configuration
GroEL/ES – quaternary structure of protein.
Gro = bacterial virus growth mutant.
cells that were resistant to bacterial virus infection.
Because bacteriaphage makes protein.
When groEL missing, bacterial virus does not replicate well.
PDI can speed up protein-folding reaction
Disulfide linkages form in the ER, as this is the oxidizing area
Different types of studying proteins:
Affinity chromatography – column has beads w/ligands attached, attractions of
proteins to ligands filters them
gel filtration/size exclusion – separates protein based on size or weight.
molecules come out first, smaller ones go inside porous beads.
ion-exchange – separates proteins by charge.
Beads attached to either an anion
exchanger or a cation exchanger.
Anion exchangers have a (+) charge, and cation
exchangers have a (-) charge.
Change pH to get protein you want out of exchangers.
Isoelectric focusing - As you change the pH, you change the ionization state of all
the aa in protein, but there is a continuum pH in which number of neg charges balance
with number of pos charges. At that pt, you have isoelectric pt, no charge.
migrates until it reaches the pH that matches its pI.
Proteins with diff pI (isoelectric pts)
will distribute differently throughout the gel.
At high pH, all proteins are neg charged.
At low pH, all proteins are pos charged. If I load the protein sample on the top here,
everything will start migrate from neg to pos. the things not neg charge will go off in
the other direction and isolate them.
It denatures proteins permanently. Heat up to 95C for 5min (very standard).
Proteins denature by heat, coat by SDS. The dodecyl suflate part binds to the protein, coats
with uniform neg charge. The larger the protein, the more SDS binds. But the charge/mass ratio
of large protein and small protein is the same. That is why SDS-PAGE is based on the protein
size not the amount of negative charges that coat the protein. Standard neg charge per mass
ratio. Remember, you are eliminating the differences in proteins, whether pos or neg charges.
They will all be uniformly neg charged. Add beta-ME (breaks disulfide linkages). So you will