principles of keeping proteins in their native state

Question 1

what is the primary protein structure?

Answer 1

the sequence of amino acids in a polypeptide chain held together with peptide bonds.

Question 2

what is the secondary protein structure?

Answer 2

alpha-helix, beta-pleated sheets held together by hydrogen bonds.

Question 3

what is the tertiary protein structure?

Answer 3

interactions between R-groups that give the protein its 3D arrangement.

Question 4

what bonds are involved in tertiary protein structure?

Answer 4

• van der waals
• ionic bonds
• hydrophobic interaction between R-groups
• hydrogen bonds
• disulphide bridges between cystine and methionine.

Question 5

what is the quaternary protein structure?

Answer 5

more than one polypeptide subunit exhibiting all previous bonds.

Question 6

what is denaturation?

Answer 6

denaturation is 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 7

what happens to quaternary protein structure during denaturation?

Answer 7

quaternary structure proteins break up into individual polypeptide subunits.

Question 8

what happens to tertiary protein structure during denaturation?

Answer 8

tertiary structure is disrupted by the breaking of bonds: ionic interactions, hydrogen bonds, van der waals, and hydrophobic interactions between R-groups.

Question 9

what happens to secondary protein structure during denaturation?

Answer 9

secondary structure proteins unravel from alpha-helix or beta-sheets, as the hydrogen bonds vibrate and break due to an increase in kinetic energy.

Question 10

what happens to primary protein structure during denaturation?

Answer 10

primary protein structure is not affected.

Question 11

how is renaturation possible?

Answer 11

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

Question 12

what conditions can result in denaturation?

Answer 12

• increase in temperature
• change in pH
• use of chemical denaturants.

Question 13

what is the effect of temperature in denaturation.

Answer 13

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 14

what is the effect of pH in denaturation?

Answer 14

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 15

what are proteases?

Answer 15

proteases breakdown proteins into short peptide fragments. (chemical denaturants)

Question 16

give an example of chemical denaturants.

Answer 16

• trypsin
• urea
• SDS

Question 17

how does trypsin work as a chemical denaturant?

Answer 17

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

Question 18

how does urea work as a chemical denaturant?

Answer 18

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

Question 19

how does SDS work as a chemical denaturant?

Answer 19

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

Question 20

what is proteolysis?

Answer 20

proteolysis is the directed degradation of a protein by proteases or by intermolecular digestion which will decrease the amount of protein present in a sample.

Question 21

what can be used to prevent proteolysis?

Answer 21

• protease inhibitor cocktail
• DTT and 2ME

Question 22

what is a protease inhibitor cocktail?

Answer 22

a protease inhibitor cocktail is designed to inhibit all protease enzymes to limit degradation.

Question 23

how are cysteine residues preserved?

Answer 23

DTT and 2ME are used to preserve cysteine residues. DTT and 2ME are reducing agents that reduce oxidation of cysteine residues, they affect the tertiary structure of a protein.

Question 24

what are common methods of storage?

Answer 24

• solution at 4ºc
• solution in 50% glycerol at -20ºc
• frozen at -20ºc or -80ºc, or in liquid nitrogen
• lyphophilized.

Question 25

how does low temperature work as a method of storage?

Answer 25

low temperatures slow down the activity of protease enzymes, thus slowing down degradation of the protein.

Question 26

how does a cryoprotectant work as a method of storage?

Answer 26

addition of glycerol which limits the formation of ice crystals.