Protein Folding and Unfolding/Denaturation Flashcards
stablizing forces
weak interactions + disulfide bond
what weak interactions are used to for stablizing forces?
hydrogen bonds, VDW interactions, intraprotein salt bridges
examples of denaturation
- detergents
- organic solutions
- extreme pH
- extreme temperature
- high ionic strength
denaturation through urea
weakens hydrogen bonds; affecting protein structure directly and indirectly
denaturation through 2-mercaptoethanol
unfolds protein via disulfide bonds
What is Tm?
aka melting temperature; temperature at which 50% of protein/DNA being analyzed is unfolded
denaturation
loss of structural integrity with accompanying loss of function
how are proteins stabilized?
by many collective weak interactions
If the Tm is higher, is the protein less or more stable?
The protein is more stable because the protein requires more heat to denature.
How to moniter the folding process in W Content?
through thermal denaturation
How to moniter the folding process in CD approach?
- CD measures the molar absorption difference βπ΄ of left- and right-circularly polarized
light: βπ΄ = π΄L β π΄R - Chromophores in the chiral environment produce characteristic signals
- CD signals from peptide bonds depend on the chain conformation
What is the famous ribonuclease refolding experiment? (experiment carried out & implications)
Protein with 8 cysteines are linked together; producing four disulfide bonds, urea and 2 mercaptothnol denatures protein by weakening hydrogen bond structure and disrupting disulfide bond, when denaturation agents are removed, the protein refold to itβs original state
because protein refolds to its original state, we know it is NOT random
MAIN CONCLUSION OF EXP:
sequence alone determines the native conformation (or the folding information is embedded in the sequence)
Is protein folding a random process? Why?
No because it folds at the lowest energy fold based on what is thermodynamically most favorable
Destabilizing forces
Primarily electrostatic interactions with solvent and conformational entropy
reduction
The hydrophobic effect is a not a true force; rather, it is a . . .
colligative property
colligative property
properties of a solution that depend on the concentration of solute particles, but not on the identity of the solute
denaturation
Loss of structural integrity with accompanying loss of function
