Protein Folding & Stability

Question 1

define: protein stability

Answer 1

How stable a protein is

held together by an accumulation of weak forces & are only marginally stable

Question 2

when are proteins not stable?

Answer 2

denatured at extremes of pH
Not stable in organic solvents as strips the essential water from the protein

organic solvents cause them to misfiled

Question 3

Folding as an equilibrium

Answer 3

fold to their energetically most favourable conformation

interaction with solvent maximal, always coating the protein
not a lot of spare bonding in folded state

Question 4

what are the forces that stabilise 3d structure?

Answer 4

hydrophobic interactions
ionic interactions
van der Waals interactions
hydrogen bonds
disulphide bonds

Question 5

hydrophobic interactions

Answer 5

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

Question 6

Physical basis for hydrophobic effect

Answer 6

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

Question 7

why do hydrophobic molecules want to be in the molecule?

Answer 7

entropically favourable for these molecules to be in the centre

Question 8

Ionic interactions

Answer 8

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

Question 9

Van der Walls interactions

Answer 9

non-covalent interactions between neutral molecules
arise from electrostatic interactions between dipoles (some sort of charge separation)

weaker than ionic interactions
many of them

Question 10

what is a dipole?

Answer 10

some sort of charge separation

Question 11

instantaneous dipole

Answer 11

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

Question 12

optimal distance

Answer 12

how far apart repulsive and attractive energy are from each other to maximise van Der Waals forces

also described as van Der Waals distance

Question 13

optimal packing

Answer 13

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

Question 14

define: hydrogen bonds

Answer 14

Electrostatic interactions between weakly acidic donor group and an acceptor atom
bears a lone pair of e-

in-between 2 electronegative atoms

Question 15

Hydrogen bonds in water

Answer 15

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

Question 16

Disulphide bonds

Answer 16

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

Question 17

what two types of bonds make the largest contribution in protein stability?

Answer 17

hydrophobic interactions
disulphide bridges

Question 18

general rules of protein folding

Answer 18

Fold spontaneously
Native conformation most energetically stable
proteins fold based on amino acid sequence
doesn’t need other factors to fold

Question 19

Step 1 of Anfinsen’s experiment

Answer 19

B-mercaptoethanol & urea removed slowly
protein refolded & correct disulphide bonds reformed
back to 100% activity

Question 20

Step 2 of Anfinsen’s experiment

Answer 20

when only B-mercaptoethanol removed, cysteines formed random disulphide bridges

Question 21

Step 3 of Anfinsen’s experiment

Answer 21

when then removing urea, only 1% of activity restored

Question 22

Conclusions: Anfinsen’s experiment

Answer 22

Urea unfolds proteins - no enzymatic activity, unfolded so no bridges

both removed together 100% activity, one then the other only 1% activity

Question 23

what does scrambled ribonuclease do?

Answer 23

Spontaneously refolds
sequence dictates structure

shows that proteins form into correct state then the disulphide bridges lock the structure into place

Question 24

Mechanism of protein folding

Answer 24

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

Question 25

Levinthal Paradox

Answer 25

time required to a protein of n residues to explore all is:
time = (no. possible conformations)^n / 10^13