7. Protein Structure

Question 1

Two important transport
proteins in humans

Answer 1

haemoglobin and cytochrome c proteins.

Question 2

ion exchange chromatography was used to do what

Answer 2

separate a mixture of these two proteins on the basis of their charge

Question 3

what chromatography did we use to determine the size of each protein

Answer 3

gel filtration/size exclusion chromatography

Question 4

how did we analyse the subunit
composition and quaternary structure of each protein

Answer 4

by running gel electrophoresis (SDS- PAGE)

Question 5

Remember that elution in ion exchange is determined by the charge interactions of the protein with the cellulose. This means that elution profile must be explained in terms of

Answer 5

electrostatic attraction/repulsion to DEAE.

Question 6

haemoglobin found where + what is its function

Answer 6

found in red blood cells
its function is to bind and transport oxygen.

Question 7

cytochrome c found where + what is its function

Answer 7

Cytochrome c is found in mitochondria, and its function is to transfer electrons in the electron transport chain.

Question 8

Due to the presence of different numbers of ionisable amino acid side chains in a protein,
what varies from protein to protein.

Answer 8

the net charge

Question 9

the net charge on a particular protein may be altered by varying what?

Answer 9

the pH of the solution.

Question 10

what is the charge on the protein at the pI

Answer 10

net neutral

Question 11

At pH below the pI, the protein will have what charge

Answer 11

positive charge (protonated)

Question 12

At pH above the pI, the protein will have what charge

Answer 12

negative charge (deprotonated)

Question 13

Ion exchange celluloses can be used to separate proteins with different charge. what do they consist of?

Answer 13

cellulose beads substituted with charged groups giving either
- a cation exchanger
- an anion exchanger

Question 14

what does a cation exchanger do

Answer 14

it is negatively charged so which will bind/exchange positively charged species. e.g. carboxymethyl (CM) cellulose

Question 15

what does a anion exchanger do

Answer 15

it is positively charged so will bind/exchange negatively charged species. e.g. diethylaminoethyl (DEAE) cellulose.

Question 16

In DEAE cellulose, the DEAE groups have a pI of ?

Answer 16

9.5

Question 17

what pH has positively charged DEAE

Answer 17

below pH 9

Question 18

two methods to elute a protein which is bound to DEAE cellulose:

Answer 18

Increase the ionic strength of the buffer.
Decrease the pH of the buffer.

Question 19

how does increasing the ionic strength of the buffer elute the protein

Answer 19

Anions in the buffer compete with the protein of interest for binding to charged groups on the DEAE.

At low ionic strength of buffer, competition between the protein and anions
is minimised, and the protein can bind to the DEAE.

Increasing the ionic strength of the buffer increases the competition, reducing the protein-DEAE interaction, and results in the elution of the protein.

Question 20

how does decreasing the pH of the buffer elute the protein

Answer 20

Lowering the pH of the buffer reduces the net excess negative charge on the surface of the protein.
When the pH of the buffer
approaches the pI of the protein, the protein will no longer contain sufficient negative charge to remain bound to the DEAE, and will elute.

Question 21

What property of proteins does gel filtration chromatography use for separation?

Answer 21

Molecular weight.

Question 22

How do large proteins behave in gel filtration chromatography?

Answer 22

They are excluded from the pores and elute first because they pass around the beads.

Question 23

How do small proteins behave in gel filtration chromatography?

Answer 23

They enter the pores and elute last because they take a longer path through the column.

Question 24

What is the function of the cellulose beads in gel filtration chromatography?

Answer 24

They form a 3D matrix with pores that separate proteins based on size.

Question 25

How can the molecular mass of an unknown protein be determined using gel filtration chromatography?

Answer 25

By comparing its elution volume to a calibration curve made with proteins of known molecular mass.

Question 26

how do we know where buffer was changed

Answer 26

it'll be the flat region between the peaks

Question 27

if a protein binds strongly to the ion exchange volume what will the elution volume be like

Answer 27

higher bc more needed to come off the volume

Question 28

Common Quaternary Structures

Answer 28

Homodimer: Two identical subunits (one band on SDS-PAGE, native mass = 2× band mass) Heterodimer: Two different subunits (two bands on SDS-PAGE, native mass = sum of bands) Tetramer: Four subunits (can be all identical or different combinations)

Question 29

On an SDS-PAGE gel: it's a monomer if

Answer 29

the native protein's mass matches the single band you see on SDS-PAGE,

Question 30

On an SDS-PAGE gel: it likely has a quaternary structure if

Answer 30

if the native protein's mass is some multiple of what you see on SDS-PAGE

Question 31

Is DEAE cellulose an anion or cation exchanger? at pH 8.8

Answer 31

an anion exchanger bc pH 8.8 is below 9 so it will be positively charged

Question 32

When do proteins elute immediately/non-bound?

Answer 32

When protein charge = column charge (repulsion) Anion exchanger (+): Proteins with pH < pI elute first Cation exchanger (-): Proteins with pH > pI elute first These form early peaks (small elution volume) No buffer change needed for elution

Question 33

How do bound proteins elute?

Answer 33

Require buffer change to elute Methods: pH shift across protein's pI Increased salt concentration Higher buffer molarity Elute in later peaks (larger elution volume) Stronger binding = later elution Example: pI between buffer pHs causes charge reversal and elution

Question 34

How to identify which peak is which protein?

Answer 34

Compare pI to buffer pH Early peaks: Non-binding proteins Later peaks: Proteins that required buffer change For anion exchanger: If pI > buffer pH: Early peak (positive, doesn't bind) If pI < buffer pH: Later peak (negative, binds) Check timing relative to buffer change