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Lecture 3

Biology 1002B Lecture Notes - Lecture 3: Macromolecular Crowding, Protein Folding, Peptide


Department
Biology
Course Code
BIOL 1002B
Professor
Tom Haffie
Lecture
3

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Lecture 3 – Protein Folding
For a protein to be functional:
oIt must fold correctly!
oPrimary sequence (polypeptide – no function because hasn’t folded
yet) as it comes o the the ribosome, must fold correctly into the
native conformation (tertiary) for it to be functional
oOccurs very fast in cells
Anfensen’s dogma:
1. Took an enzyme from e-coli and measured the activity of the enzyme
(catalytic potential of the enzyme – how good it is at driving a reaction)
2. Added urea, denaturing the protein, making the protein unfold
3. Urea is very polar, and competes with the amino acids in the protein
for hydrogen bonds  disrupts normal hydrogen bonding because it is
polar itself (lots of positive and negative charges to contribute to
bonding)
Common denaturant
4. 0 enzyme activity – reaction catalyzing it drops to 0 in presence of
molar concentrations of urea
5. Removed urea and replace the urea with buer
6. Refolds and about 90% activity is back  go from native 100% active to
native >90% active  will keep folding into exact same tertiary shape
Protein folding doesn’t need anything except the primary sequence of the
protein itself
oSpontaneous, very fast, in milliseconds
oTertiary structure/native structure the protein acquires is dependent
solely on primary sequence
No ATP required
Primary sequence has all the information required for the protein
to get to the <nal tertiary structure
MACROMOLECULAR CROWDING
All of this occurs in-vitro (in a test tube): concentrations of protein are low but
in an actively growing cell, protein concentrations are 3000x higher  referred
to as macromolecular crowding
Macromolecular crowding interferes with protein folding
oIf a polypeptide wants to fold and produce an H-bond but other
polypeptides get in the way with dierent side groups
oSo many polypeptides in a solution that proteins start folding together
oThings don’t work in-vivo
oNot one polypeptide, but many
Macromolecular crowding is an issue within an intact cell
For many proteins, they need help to fold  help comes from another class of
proteins called chaperones

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CHAPERONES
Assist a protein in folding
Unfolded protein goes in and then folded protein comes back out
Don’t aect the component of tertiary structure  not involved in that
Requires energy (ATP)
Human genome codes for 100s of dierent chaperones
o1 chaperone can’t fold many types of proteins
Heat shock proteins is a type of chaperone
oHeat shock (heat being denaturant – not folding under high
temperature)
oHeat shock proteins facilitate proper protein folding
WHAT DRIVES PROTEIN FOLDING? LEVINTHAL PARADOX
In low enough concentrations, when there is no interference with other
polypeptides, the protein will spontaneously acquire correct 3D shape
A polypeptide will always acquire the exact same tertiary shape
Levinthal Paradox:
oExample: small protein of 100 amino acids
If look at an amino acid with a peptide bond on each side, amino
acid can acquire multiple conformations
If it can assume 3 dierent conformations over a billion dierent
structures
Converting between structures would have over a billion years
oWhen a polypeptide folds, it doesn’t try out every possible
conformation (it would take too long)
Levinthal paradox explains how proteins fold so fast –they do this by not
checking out every possible conformation
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