Lecture 7 concepts: Protein folding II Flashcards
Protein stability created by
Hydrophobic effect: keeps hydrophobic r chains on the inside and hydrophilic r chains on the outside through the release of water molecules from the nonpolar molecules (water disperses, thus increasing entropy of surroundings)
Disulfide linkages
“Ligameric structures” Subunit association: proteins associate with ligameric structures (quartenary structure) ie subunit associations: example- three polypeptide chains associating with one another.
“fine tuning” bonds
- secondary bonding such as hydrogen bonds and disulfide bonds, ionic bonds and van der waals interactions
Symmetry: why C3 C4 etc?
important for protein stability
you can turn them around that number of times and see the same thing
“Screw symmetry”
actin helical structures/ microtubules
UPR
Unfolding Protein Response is a cellular response launched by the ER in response to UPS: halts elongation and degrades peptide. if this doesn’t work in clicks into apoptosis
Protein Denaturation (Conditions)
Heat, pH, agitation
Heat: brings the hydrophobic interiors of egg whites to the outside, causing them to aggregate (making white color)
pH: breaks salt bonds and denatures
agitation: mechanical energy denatures proteins (frothing eggs for example)
Protein Denaturation (Chemicals)
1) detergents
2) chaotropic agents (little molecules/metals that can disrupt bonds: urea and guanadinium chloride)
3) organic solvents (alcohol)
Denaturing a protein: if you look at sample of denaturing protein at the midway point, what do you see?
50% denatured, 50% undenatured. Either/or, highly cooperative. Midway point is called “TN”
“TN”
midway point of denatured proteins
Methods of analyzing denatured proteins and related terms
1) turbidity (shaking around)
2) circular dichroism (CD: looking at secondary structure of protein itself, % of alpha helice and beta strand)
3) UV absorbance (aromatic, because they absorb
a) microenvironment will be different in the folded/unfolded state
4. biological activity (do these proteins have any activity?)
Chaotropic Agents (name the one beginning with a G)
Guanidine (deprotonated)—–> Guanidinium (protonated)
Can reversibly bind a proton
Chaotropic Agents II (name the one beginning with U)
Urea reducing agent, breaks down disulfide bonds
Chaotropic bonds III (name the one beginning with b)
beta-mercaptoethanol, reducing again, completely breaks down disulfide bonds
Where would you find chatropic agents? Where would they work? How?
They reduce disulfide bonds, so you would find them on extracellular proteins (because DSulfide bonds can withstand oxidative environments (already are oxidized)
They would work anywhere with disulfide bonds
They work through reduction: adding an H plus two electrons to their target
Before you perform electrophoresis you would
reduce disulfide bonds
Accessory Proteins: PDI
protein disulfide isomerases: fixes misses up disulfide bonds from misbonded proteins intended for export
they break the wrong ones and reform the correct ones
Accessory Proteins: PPI
Proline has trans/cis, but energetically they are the same. PPI corrects the cis-trans depending on which it’s supposed to be
“protein cis-trans prolyl isomerases”
Molecular chaperones: HSP 70 and 40
Reverse misfolds, ATP drive
participation in synthesizing new proteins
participate in delivery of proteins to mitochondria
HSP 90
signal transduction proteins involved in folding IUP (intrinsically unstructured proteins)
Nucleoplasmins
Involved in nucleus sole formation, ribogenesis as well
Small-HSPs
25-50 exist depending on cell type
prevent aggregation of misfolded proteins,
work with the HSP 70 and HSP 40
Chaperonins
basket like structure proteins that “catch” misfolded proteins and fix them
Two Models (mechanisms) of Protein Folding
Passive Mechanism: unfolded protein is placed into a “cage” (protein) and allowed to reassemble itself by itself
Iterative Annealing: an accessory protein interacts with the unfolded protein to repair it
Iterative Annealing
an accessory protein interacts with the unfolded protein to repair it: the inner part of the “basket” actually interacts with the unfolded protein. It’s an “active model”
Passive Mechanism
“cage” model
unfolded protein is placed into a case and allowed to reassemble itself
4 Steps of either the Iterative or the CAGE model
1) unfolded protein (UP) binds to apical domain of cage protein
a) the hydrophobic domain most likely
2) the GroES snaps shut
a) requires ATP input
3) Protein allowed to find itself OR the cage interacts with the protein to assist it
a) release of energy
4)GroES comes off and out goes the newly folded protein
Presence of methionine sulfoxide means
Protein is worn out, thiol group can be reduced
Chaperonins: name some
GroEs/El, group 1, hsp 60/10 mitochondrial: found in bacteria
CCTV group II found in eukaryotic cytosol
Both are dihedral
Where might you find chaperonins?
Places where proteins are being broken down, muscle cells for example because your fibrous proteins are being broken down and reformed
C7 is what
Cap to the group I chaperonins
