C. PROTEIN CHEMISTRY 3

Question 1

post-translational modifications of proteins

Answer 1

• phosphorylation of hydroxyl groups Ser, Thr or Tyr
• can or can’t make interactions due to added charge
• ie:
  1. Tyr phosphorylation of receptor
  Tyrosine kinase receptors
  Phosphorylations are key to signalling pathways in the cell

Question 2

what are the 4 types of post translational modifications

Answer 2

1. Glycosylation
   - Attachment of sugar
   moieties to Ser, Thr or Asn residues, can alter solubility (more stable)
2. Hydroxylation
   - Hydroxyl group (OH) added
   to Pro or Lys residues, can alter hydrogen
   bonding
3. Methylation: Methyl groups can be added
   to nitrogen or oxygen atoms of amino acid side chains: added hydrophobic group
4. Disulfide bond formation between two cysteines, typically renders proteins more stable: additional covalent linkage

Question 3

if a protein has many basic side chains, what is the overall charge at physiological pH

Answer 3

net +ve charge

Question 4

if a protein has many acidic side chains, what is the overall charge at physiological pH

Answer 4

net -ve charge

Question 5

if a protein has acidic and basic side chains, what is the overall charge at physiological pH

Answer 5

whichever one is more frequent

Question 6

what determines the protein’s state of ionisation

Answer 6

• amino acids
• pH of the solution environment

Question 7

what is the isoelectric point (pI)

Answer 7

pH at which the molecule or surface carries no net electrical charge

Question 8

what are the 2 consequences of the pI

Answer 8

1. doesn’t migrate in an electric field
2. protein is least soluble

normally pH range of 5.5-8

Question 9

what is the influence of pI on solubility

Answer 9

• Insulin glargine is a long-acting insulin that contains two extra arginine residues (basic) at the end of the B chain
• increases isoelectric point of insulin (normally ~5.4) to 6 ish and alters the solubility, more soluble in the acidic
  conditions (pH 4) used in the formulation, but
  less soluble upon injection (encounters pH 7.4)

Question 10

what is separation of different proteins based on

Answer 10

• charge
• hydrophobicity
• solubility
• size

Question 11

what is gel electrophoresis

Answer 11

• for separation and analysis of macromolecules (DNA and proteins)
• separated according to their size and charge
• electrophoresed within a matrix or “gel”
• in an electric field charged molecules migrate toward either the positive or
  negative pole according to their charge

Question 12

where do anions move towards

Answer 12

the anode (+ve)

Question 13

where do cations move towards

Answer 13

the cathode (-ve)

Question 14

what are the forces of attraction

Answer 14

• size of charge
• size of electric field

Question 15

forces of retardation

Answer 15

• friction
• repulsion in medium

Question 16

what type of species have high mobility

Answer 16

small, highly charged species

Question 17

what type of species have low mobility

Answer 17

large, minimally charged species

Question 18

how does the structure the analyte affect migration

Answer 18

• analytes must be charged/have a charge induced
• contain acidic or basic functional groups
• ionisation dependent on pKa of functional groups and pH of electrolyte
• pH>pKa = deprotonated

Question 19

what is native polyacrylamide gel electrophoresis (PAGE)

Answer 19

• ‘native’ structure of protein is maintained during electrophoresis
• separation according to size and charge

Question 20

how does size affect separation

Answer 20

• acrylamide gels serve as a size-selective sieve during separation
• as proteins move through a gel in response to an electric field, the smaller molecules travel more rapidly

Question 21

how does charge affect separation

Answer 21

• highly negatively charged molecules will migrate faster than less negatively charged molecules towards the anode
• these two effects in combination can mean that a highly negatively charged larger molecule can migrate faster than a less negatively charged smaller molecule

Question 22

applications of native PAGE electrophoresis

Answer 22

• assess quaternary structure (oligomerisation state) of a protein
• homogeneity of a pure protein sample

ie - insulin, might see a band for dimer and a band for the hexamer

Question 23

what is SDS-polyacrylamide gel electrophoresis

Answer 23

• ‘native’ structure of proteins is NOT maintained during electrophoresis: proteins are denatured
• separation according to SIZE
• proteins are denatured by heat and the addition of the detergent SDS (Sodium dodecyl sulfate) prior to electrophoresis

Question 24

sodium dodecyl sulfate trick

Answer 24

• surfactant = polar to aq sol and non-polar to oil on top
• emulsification by the detergent (SDS) gives proteins a net negative charge
• different proteins in the same SDS solution are imparted with approximately the same charge to mass ratio, so will predominantly migrate according to size alone

Question 25

what are the 2 ways to detect proteins post-electrophoresis

Answer 25

1. Coomassie Brilliant blue dye staining - dye needs to have conjugated double bonds to absorb visible light and hence show colour 2. Western blot (protein immunoblot) - transfer of gel contents onto a membrane and detection via labelled antibodies

Question 26

how to measure protein concentrations

Answer 26

UV absorption - a dissolved substance will absorb light of specific wavelengths characteristic of that substance - Extinction coefficient/Lambert Beer Law

Question 27

advantages of UV absorption

Answer 27

- no additional reagents or incubations are required - no protein standard need to be prepared - the assay does not consume the protein - the relationship of absorbance to protein concentration is linear

Question 28

disadvantages of UV absorption

Answer 28

- any non-protein component in the solution that absorbs ultraviolet light will interfere with the assay

Question 29

what are the absorbance maximums

Answer 29

- proteins in solution absorb ultraviolet light with absorbance maxima at 280 nm and 200 nm - 280 nm: mainly due to amino acids with aromatic rings - 200 nm: mainly due to peptide bonds - secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum

Question 30

what are the 2 colorimetric methods for measuring protein concentrations

Answer 30

1. Bradford assay: dye based 2. BCA protein assay: copper based A standard curve with samples of known protein concentrations is created, and the concentration of the unknown protein is determined from the curve

Question 31

Bradford assay: dye based

Answer 31

- Coomassie Brilliant Blue dye binds to proteins in acidic solution (via electrostatic and van der Waals bonds) resulting in a shift of the absorption maximum of the dye from 465 to 595 nm

Question 32

BCA protein assay: copper based

Answer 32

- principle: reduction of Cu2+ to Cu1+ by protein in an alkaline medium with colorimetric detection of the cuprous cation (Cu1+) by bicinchoninic acid (BCA) - intense purple-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion - the BCA/copper complex exhibits a strong linear absorbance at 562 nm with increasing protein concentrations

Question 33

advantage of BCA Protein Assay: copper based

Answer 33

- unlike the Coomassie dye-binding methods, the peptide backbone also contributes to colour formation, helping to minimize variability caused by protein compositional difference