C. PROTEIN CHEMISTRY 3 Flashcards
post-translational modifications of proteins
- 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
what are the 4 types of post translational modifications
- Glycosylation
- Attachment of sugar
moieties to Ser, Thr or Asn residues, can alter solubility (more stable) - Hydroxylation
- Hydroxyl group (OH) added
to Pro or Lys residues, can alter hydrogen
bonding - Methylation: Methyl groups can be added
to nitrogen or oxygen atoms of amino acid side chains: added hydrophobic group - Disulfide bond formation between two cysteines, typically renders proteins more stable: additional covalent linkage
if a protein has many basic side chains, what is the overall charge at physiological pH
net +ve charge
if a protein has many acidic side chains, what is the overall charge at physiological pH
net -ve charge
if a protein has acidic and basic side chains, what is the overall charge at physiological pH
whichever one is more frequent
what determines the protein’s state of ionisation
- amino acids
- pH of the solution environment
what is the isoelectric point (pI)
pH at which the molecule or surface carries no net electrical charge
what are the 2 consequences of the pI
- doesn’t migrate in an electric field
- protein is least soluble
normally pH range of 5.5-8
what is the influence of pI on solubility
- 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)
what is separation of different proteins based on
- charge
- hydrophobicity
- solubility
- size
what is gel electrophoresis
- 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
where do anions move towards
the anode (+ve)
where do cations move towards
the cathode (-ve)
what are the forces of attraction
- size of charge
- size of electric field
forces of retardation
- friction
- repulsion in medium
what type of species have high mobility
small, highly charged species
what type of species have low mobility
large, minimally charged species
how does the structure the analyte affect migration
- 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
what is native polyacrylamide gel electrophoresis (PAGE)
- ‘native’ structure of protein is maintained during electrophoresis
- separation according to size and charge
how does size affect separation
- 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
how does charge affect separation
- 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
applications of native PAGE electrophoresis
- 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
what is SDS-polyacrylamide gel electrophoresis
- ‘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
sodium dodecyl sulfate trick
- 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
what are the 2 ways to detect proteins post-electrophoresis
- Coomassie Brilliant blue dye staining
- dye needs to have conjugated double bonds to absorb visible light and hence show colour - Western blot (protein immunoblot)
- transfer of gel contents onto a membrane and detection via labelled antibodies
how to measure protein concentrations
UV absorption
- a dissolved substance will absorb light of specific wavelengths characteristic of that substance
- Extinction coefficient/Lambert Beer Law
advantages of UV absorption
- 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
disadvantages of UV absorption
- any non-protein component in the solution that absorbs ultraviolet light will interfere with the assay
what are the absorbance maximums
- 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
what are the 2 colorimetric methods for measuring protein concentrations
- Bradford assay: dye based
- 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
Bradford assay: dye based
- 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
BCA protein assay: copper based
- 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
advantage of BCA Protein Assay: copper based
- unlike the Coomassie dye-binding methods, the peptide backbone also contributes to colour formation, helping to minimize variability caused by protein compositional difference
