OC1 - general principles of denaturation

Question 1

what is denaturation?

Answer 1

the major change in a proteins structure by application of an external stress or compound
when a protein is denatured the shape of the active site is altered which prevents a substrate from binding and completing its function, rendering the protein non-functional.

Question 2

primary protein structure

Answer 2

the sequence of amino acids held together by peptide bonds
denaturation > not affected

Question 3

secondary protein structure

Answer 3

alpha-helix, beta-pleated sheets held together by hydrogen bonds
denaturation > unravel from alpha-helix or beta-sheets as the hydrogen bonds vibrate and break due to an increase in kinetic energy

Question 4

tertiary protein structure

Answer 4

interactions between R-groups that give the protein its 3D arrangement
bonds: Van Der Waals, ionic bonds, hydrophobic interactions between R-groups, hydrogen bonds, disulphide bridges between cysteine and methionine.
denaturation > disrupted by the breaking of bonds : ionic interactions, hydrogen bonds, Van Der Waals, hydrophobic interactions between R-groups.

Question 5

quaternary protein structure

Answer 5

more than one polypeptide subunit exhibiting all previous bonds
denaturation > break up into individual polypeptide subunits

Question 6

renaturation

Answer 6

denaturation can be reversible. renaturation is possible as all the information for the protein is contained in the primary structure of protein.

Question 7

temperature

Answer 7

proteins have an optimum temperature of 37ºC - 40ºC.
an increase in temperature above the optimum will provide too much kinetic energy, which causes the atoms to vibrate making them thermally unstable.
this increase in energy overcomes the hydrogen bonds holding the proteins active site together, changing its shape, therefore rendering the protein non-functional

Question 8

pH

Answer 8

a change in pH will alter the acidic and basic groups on amino acid side chains in a proteins active site by altering the equilibrium point of ionisation, this renders the protein inactive but maintains its 3D arrangement.

Question 9

chemical denaturants

Answer 9

proteases breakdown proteins into short peptide fragments
trypsin, urea, SDS

Question 10

trypsin

Answer 10

cleaves the polypeptide chain at the carboxyl side of lysine and arginine, except when either is followed by a proline
should be stored at very low temperatures to prevent autolysis

Question 11

urea

Answer 11

denatures proteins by decreasing the hydrophobic effect, and by directly bonding to the amide groups of the protein via hydrogen bonding.

Question 12

SDS

Answer 12

SDS (sodium dodecyl sulphate) is a detergent with a hydrophobic tail which will dissolve hydrophobic molecules, and a negatively charged hydrophilic head.
if a cell is incubated with SDS its membranes will dissolve, all the proteins will be solubilised by the detergent and will be given a negative charge. the protein will retain only its primary structure, and will have a negative charge which means all the proteins will migrate towards the positive pole when placed in an electric field.