Protein Folding & Stability Flashcards
define: protein stability
How stable a protein is
held together by an accumulation of weak forces & are only marginally stable
when are proteins not stable?
denatured at extremes of pH
Not stable in organic solvents as strips the essential water from the protein
organic solvents cause them to misfiled
Folding as an equilibrium
fold to their energetically most favourable conformation
interaction with solvent maximal, always coating the protein
not a lot of spare bonding in folded state
what are the forces that stabilise 3d structure?
hydrophobic interactions
ionic interactions
van der Waals interactions
hydrogen bonds
disulphide bonds
hydrophobic interactions
have non-polar side chains
like to be away from water, so are present inside globular proteins
make the biggest contribution to folding
want to be away from water
no charges on r groups, no OH groups
Physical basis for hydrophobic effect
leads to clathrate- cage encoding every droplet
water encages as choice predetermined
entropically favourable for droplets to join to liberate some of the waters encapsulated
why do hydrophobic molecules want to be in the molecule?
entropically favourable for these molecules to be in the centre
Ionic interactions
salt bridges
electrostatic interaction occurs between + and - charged amino acid residues
removal of salvation shell needed to form ionic bond
desolate the ions so can bring them closer together
Van der Walls interactions
non-covalent interactions between neutral molecules
arise from electrostatic interactions between dipoles (some sort of charge separation)
weaker than ionic interactions
many of them
what is a dipole?
some sort of charge separation
instantaneous dipole
by chance and fora brief instance
e- clouds on either side of orbitals
other molecule attracted by e- to the +
known as London dispersion forces
form when folded or unfolded
optimal distance
how far apart repulsive and attractive energy are from each other to maximise van Der Waals forces
also described as van Der Waals distance
optimal packing
interaction between permanent dipoles
some atoms are more electronegative than needed
can induce charge separation and give rise to van Der Waals
example: permanent dipole on C=O will always attract another carbonyl group
define: hydrogen bonds
Electrostatic interactions between weakly acidic donor group and an acceptor atom
bears a lone pair of e-
in-between 2 electronegative atoms
Hydrogen bonds in water
any molecule that can H bond to itself can also H bond to water
H bonds formed and dissolved in water are relatively weak
unfolded H bonds formed between protein and solvent, break 2 H bonds allowing for H bond to form when folded
Disulphide bonds
covalent binds between 2 cysteine amino acids and protein chain
only covalent bond found to hold the protein structure
not very common & require oxidising conditions for the bonds to form
cysteine has to be present for bonds to form in the amino acid
what two types of bonds make the largest contribution in protein stability?
hydrophobic interactions
disulphide bridges
general rules of protein folding
Fold spontaneously
Native conformation most energetically stable
proteins fold based on amino acid sequence
doesn’t need other factors to fold
Step 1 of Anfinsen’s experiment
B-mercaptoethanol & urea removed slowly
protein refolded & correct disulphide bonds reformed
back to 100% activity
Step 2 of Anfinsen’s experiment
when only B-mercaptoethanol removed, cysteines formed random disulphide bridges
Step 3 of Anfinsen’s experiment
when then removing urea, only 1% of activity restored
Conclusions: Anfinsen’s experiment
Urea unfolds proteins - no enzymatic activity, unfolded so no bridges
both removed together 100% activity, one then the other only 1% activity
what does scrambled ribonuclease do?
Spontaneously refolds
sequence dictates structure
shows that proteins form into correct state then the disulphide bridges lock the structure into place
Mechanism of protein folding
The protein has a specific rout of folding
it cannot explore all possibilities then go through the stages of structure
proteins must fold to their native conformations by directed pathways rather than random search methods
Levinthal Paradox
time required to a protein of n residues to explore all is:
time = (no. possible conformations)^n / 10^13
