Quiz 6

Question 1

What are some methods of quantifying protein concentration?

Answer 1

Absorbance at 280nm
Bradford protein assay
Lowery method
BCA assay

Question 2

Describe simple optical density measurement

Answer 2

Absorbance at 280nm (aromatic ring side chains such as trp and tyr) or 205nm (peptide bond) provides a rough approximation of protein concentration
If a protein sequence is known the theoretical extinction coefficient at 280nm can be estimated using the equation
E280nm(Mcm) = (#trp)(5500) + (#tyr)(1490) + (#cys)(125)

Question 3

How much does a typical A280 unit of proteins equal?

Answer 3

1 mg/mL

Question 4

What is the Bradford assay?

Answer 4

Relies on coomassie brilliant blue G-250
Negatively charged due binds to positively charged amino acids (lysine, arginine, histidine)
Shifts absorbance from 465 to 595nm
Detergent can result in high background with this assay

Question 5

What is the lowery assay?

Answer 5

Copper ions react with peptide bonds under alkaline conditions
Oxidation of aromatic amino acid residues (tyrosine and tryptophan)
Requires folin-ciocalteu reagent (a mixture of phosphotungstic acid and phisphomolybdic acid) which becomes reduced upon oxidation of amino acids
Concentration of reduced folin-ciocalteu reagent is measured at 750nm

Question 6

Describe the biuret assay

Answer 6

Reagent is made of sodium hydroxide, hydrated copper (II) sulphate and potassium sodium titrate
Copper ion firms violet coloured coordination complexes with the nitrogen in the peptide bind in an alkaline solution
Potassium sodium tartrate is assed to chelate and thus stabilize the cupric ions
Purple colour has an absorption at 540nm

Question 7

Describe enzyme purification

Answer 7

As enzymes become more pure, their specific activity (total activity/total protein(mg)) increases
Percent recovery decreases with each step of purification
Protein purity increases with each step

Question 8

Give an example of a protein purification scheme

Answer 8

Crude extract
Salt precipitation 
Ion exchange chromatography
Molecular size chromatography
Immunoaffinity chromatography

Question 9

Describe how the funnel helps in protein folding

Answer 9

Represents a free energy surface or landscape for the folding process
Rapid and reversible formation of local secondary structures is followed by a slower phase in which establishment of partially folded intermediates leads to the final tertiary structure
Follows the rules of thermodynamics

Question 10

What are the three pathways involved in protein folding after synthesis?

Answer 10

A) HSP- and chaperone-independent folding
B) HSP- dependent folding
C) HSP-dependent folding with chaperone proteins (such as GroES-GroEL complex in e.coli

Question 11

Describe the groES-groEL complex in e.coli?

Answer 11

An unfolded (U) or partial folded (I) polypeptide binds to hydrophobic patches on the apical ring of alpha7-subunit of GroEL followed by ATP binding, groES association and folding of the protein to its native structure (N)
14 ATP = 7ADP + 7Pi

Question 12

Describe the folding of secreted proteins

Answer 12

1. Signal recognition particle binds to signal sequence on translating ribosome
2. Signal recognition particle binds to signal receptors while Bip binds to translocon
3. Signal recognition particle unbinds and translating ribosome remains bound to translocon
4. Protein travels from ribosome through translocon and BiP, clipping signal sequence
5. Signal sequence clipped and secreted protein folds within the ER lumen

Question 13

What are some diseases of protein misfolding?

Answer 13

Alzheimer’s (amyloid peptide)
Familial amyloidotic polyneuropathy (transthyretin)
Cancer (p53)
Creutzfeldt-Jakob disease (prion)
Hereditary emphysema (alpha antitrypsin)
Cystic fibrosis (CFTR)

Question 14

Describe the measurement of protein unfolding by tryptophan fluorescence

Answer 14

Phe, tyr, trp all have inherent fluorescence as measured at 340:320nm
In hydrophobic environment, tyr and trp have high fluoresence
As he protein unfolds, exposure to a hydrophilic environment causes decreased fluorescence
As protein unfolds, hydrophobic regions become exposed
SYPRO orange binds to hydrophobic regions and fluoreses
Causes SYPRO orange to dissociate

Question 15

Describe SYPRO orange dye

Answer 15

Excitation=472nm
Emission=520nm
Read at ex.=485 and em.=625 through bio-rad filters
Can also stain protein gels 
Mass spectrometer comparable

Question 16

What are some other methods of measuring protein unfolding?

Answer 16

Circular dichroism
Differential scanning calorimetry
Isothermal titration calorimetry

Question 17

What are some different ways a protein can unfold?

Answer 17

1. Native protein becomes transient intermediate using GdHCl or urea then completely denatures with irriversible activity loss
2. Native protein becomes intermediate state with structure relaxation and 35% activity loss in basic conditions then undergoing dimer seperation
3. Native protein undergoes intit unfolding at high temperature then causes aggregation